Prevotella intermedia boosts OSCC progression through ISG15 upregulation: a new target for intervention.

PURPOSE: Periodontitis-associated bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are closely linked to the risk of oral squamous cell carcinoma (OSCC). Emerging studies have indicated that another common periodontal pathogen, Prevotella intermedia (P. intermedia), is enriched in OSCC and could affect the occurrence and progression of OSCC. Our aim is to determine the effects of P. intermedia on the progression of OSCC and the role of antibiotics in reversing these effects. METHODS: In this study, a murine xenograft model of OSCC was established, and the mice were injected intratumorally with PBS (control group), P. intermedia (P.i group), or P. intermedia combined with an antibiotic cocktail administration (P.i + ABX group), respectively. The effects of P. intermedia and ABX administration on xenograft tumor growth, invasion, angiogenesis, and metastasis were investigated by tumor volume measurement and histopathological examination. Enzyme-linked immunosorbent assay (ELISA) was used to investigate the changes in serum cytokine levels. Immunohistochemistry (IHC) was adopted to analyze the alterations in the levels of inflammatory cytokines and infiltrated immune cells in OSCC tissues of xenograft tumors. Transcriptome sequencing and analysis were conducted to determine differential expression genes among various groups. RESULTS: Compared with the control treatment, P. intermedia treatment significantly promoted tumor growth, invasion, angiogenesis, and metastasis, markedly affected the levels of inflammatory cytokines, and markedly altered M2 macrophages and regulatory T cells (Tregs) infiltration in the tumor microenvironment. However, ABX administration clearly abolished these effects of P. intermedia. Transcriptome and immunohistochemical analyses revealed that P. intermedia infection increased the expression of interferon-stimulated gene 15 (ISG15). Correlation analysis indicated that the expression level of ISG15 was positively correlated with the Ki67 expression level, microvessel density, serum concentrations and tissue expression levels of inflammatory cytokines, and quantities of infiltrated M2 macrophages and Tregs. However, it is negatively correlated with the quantities of infiltrated CD4+ and CD8+ T cells. CONCLUSION: In conclusion, intratumoral P. intermedia infection aggravated OSCC progression, which may be achieved through upregulation of ISG15. This study sheds new light on the possible pathogenic mechanism of intratumoral P. intermedia in OSCC progression, which could be a prospective target for OSCC prevention and treatment.

Heat-killed Prevotella intermedia promotes the progression of oral squamous cell carcinoma by inhibiting the expression of tumor suppressors and affecting the tumor microenvironment.

BACKGROUND: Oral microbial dysbiosis contributes to the development of oral squamous cell carcinoma (OSCC). Our previous study showed that Prevotella intermedia (P. intermedia) were enriched in the oral mucosal surface, plaque, and saliva of patients with OSCC. Intratumoral microbiome could reshape the immune system and influence the development of various tumors. However, the invasion status of human OSCC tissues by P. intermedia and the pathway through which intratumoral P. intermedia potentiates tumor progression remain unexplored. METHODS: P. intermedia in human OSCC or normal tissues was detected by FISH. A mouse OSCC cell line SCC7 was adopted to investigate the effects of heat-killed P. intermedia treatment on cell proliferation, invasion, and cytokine release by using CCK-8 assay, transwell invasion assay and ELISA. Moreover, we established a mouse transplanted tumor model by using SCC7 cells, injected heat-killed P. intermedia into tumor tissues, and investigated the effects of heat-killed P. intermedia on tumor growth, invasion, cytokine levels, immune cell infiltrations, and expression levels by using gross observation, H&E staining, ELISA, immunohistochemistry, mRNA sequencing, and transcriptomic analysis. RESULTS: Our results indicated that P. intermedia were abundant in OSCC and surrounding muscle tissues. Heat-killed P. intermedia promoted SCC7 cell proliferation, invasion and proinflammatory cytokine secretions, accelerated transplanted tumor growth in mice, exacerbate muscle and perineural invasion of OSCC, elevated the serum levels of IL-17A, IL-6, TNF-α, IFN-γ, and PD-L1, induced Treg cells M2 type macrophages in mouse transplanted tumors. The data of transcriptomic analysis revealed that heat-killed P. intermedia increased the expression levels of inflammatory cytokines and chemokines while reduced the expression levels of some tumor suppressor genes in mouse transplanted tumors. Additionally, IL-17 signaling pathway was upregulated whereas GABAergic system was downregulated by heat-killed P. intermedia treatment. CONCLUSIONS: Taken together, our results suggest that P. intermedia could inhibit the expression of tumor suppressors, alter the tumor microenvironment, and promote the progression of OSCC.

A multi-center cross-sectional study of 1495 Chinese oral lichen planus patients.

OBJECTIVE: To explore the clinical epidemiological characteristics of oral lichen planus (OLP) and risk factors for erosive/ulcerative OLP. MATERIALS AND METHODS: Patients diagnosed with OLP from 11 different hospitals were included in the study. Descriptive statistical methods were used to explore the clinical epidemiological characteristics and logistic regression, sensitivity analysis, and subgroup analysis were utilized to explore the risk factors for erosive/ulcerative OLP. RESULTS: The average age of patients was 49.2 ± 13.3 years, and 61.4% of the patients were women. The ratios of patients with reticular, hyperemic/erythematous, and erosive/ulcerative lesions were 47.9%, 27.8%, and 24.2%, respectively. Analysis of risk factors for erosive/ulcerative OLP identified the following variables: age, course of disease of 12 months or more, II°-III° dental calculus, hypertension, diabetes, and heart disease, as well as regions of habitation. Subgroup analysis showed significant differences in risk factors for erosive/ulcerative OLP in patients with and without risk behaviors. CONCLUSION: The clinical epidemiological characteristics of patients with OLP in the Chinese population in this study are basically consistent with existing reports in developed countries. And we identified clinical characteristics associated with erosive/ulcerative OLP through clinical epidemiological analysis.

Attenuation of NLRP3 Inflammasome by Cigarette Smoke is Correlated with Decreased Defense Response of Oral Epithelial Cells to Candida albicans.

BACKGROUND: It is well recognized that both smoke and Candida infection are crucial risk factors for oral mucosal diseases. The nucleotide-binding domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and its downstream effectors, interleukin (IL)-1ß and IL-18, are pivotal to the host defense against Candida and other pathogens. METHODS: The present study was designed to explore the effects of cigarette smoke and C. albicans on the NLRP3 inflammasome and its downstream signal pathway via in vitro cell model. Oral epithelial cells (Leuk-1 cells) were exposed to cigarette smoke extract (CSE) for 3 days and/or challenged with C. albicans. RESULTS: Microscopically, Leuk-1 cells exerted a defense response to C. albicans by markedly limiting the formation of germ tubes and microcolonies. CSE clearly eliminated the defense response of Leuk-1 cells. Functionally, CSE repressed NLRP3 inflammasome, and IL-1ß and IL-18 activation induced by C. albicans in Leuk-1 cells. CONCLUSION: Our results suggested that in oral epithelial cells, the NLRP3 inflammasome might be one of the target pathways by which CSE attenuates innate immunity and leads to oral disorders.

The potential roles of cigarette smoke-induced extracellular vesicles in oral leukoplakia.

BACKGROUND: The onset of oral leukoplakia (OLK), the most common oral lesion with a high risk of malignant transformation, is closely associated with the exposure of cigarette smoke. Cigarette smoke is a complicated mixture of more than 4500 different chemicals including various oxidants and free radical, which contributes to the onset of immune and inflammatory response or even carcinogenesis. Recent studies have proved that the exposure of cigarette smoke leads to the onset and aggravation of many diseases via significantly changed the production and components of extracellular vesicles. The extracellular vesicles are membrane-enclosed nanosized particles secreted by diverse cells and involved in cell-cell communication because of their ability to deliver a number of bioactive molecules including proteins, lipids, DNAs and RNAs. Getting insight into the mechanisms of extracellular vesicles in regulating OLK upon cigarette smoke stimulation contributes to unravel the pathophysiology of OLK in-depth. However, evidence done on the role of extracellular vesicles in cigarette smoke-induced OLK is still in its infancy. MATERIALS AND METHODS: Relevant literatures on cigarette smoke, oral leukoplakia and extracellular vesicles were searched in PubMed database. CONCLUSIONS: In this review, we summarize the recent findings about the function of extracellular vesicles in the pathogenesis of cigarette smoke-induced diseases, and to infer their potential utilizations as diagnostic biomarkers, prognostic evaluation, and therapeutic targets of OLK in the future.

Oral squamous cell carcinoma-derived EVs promote tumor progression by regulating inflammatory cytokines and the IL-17A-induced signaling pathway.

BACKGROUND: Inflammatory cytokines in the tumor microenvironment (TME) contribute to tumor growth, proliferation, and invasion, and tumor-derived extracellular vesicles (EVs) act as critical "messengers" of communication in the tumor microenvironment. The effects of EVs derived from oral squamous cell carcinoma (OSCC) cells on tumor progression and the inflammatory microenvironment are still unclear. Our study aims to investigate the role of OSCC-derived EVs in tumor progression, the imbalanced TME, and immunosuppression and their effect on the IL-17A-induced signaling pathway. METHODS: EVs were isolated from the supernatant of a mouse OSCC cell line, SCC7. The effects of SCC7-EVs and the EV release-specific inhibitor GW4869 on the proliferation and migration of SCC7 cells were investigated in vitro by using CCK-8 and scratch wound healing assays. RT-qPCR and ELISA were performed to examine the alterations in cytokine levels. Then, a mouse xenograft model of OSCC was established by submucosal injection of SCC7 cells with or without SCC7-EV and GW4869 treatment. The effects of GW4869 and SCC7-EVs on xenograft tumor proliferation and invasion were investigated by tumor volume determination and histopathological examination. ELISA was used to investigate the changes in serum cytokine levels. Immunohistochemistry was adopted to analyze the alterations in the levels of inflammatory cytokines, immune factors, and crucial molecules in the IL-17A signaling pathway. RESULTS: SCC7-derived EVs increased the supernatant and serum levels of IL-17A, IL-10, IL-1ß, and PD-L1, while GW4869 decreased those of TNF-α and IFN-γ. SCC7-EV treatment significantly increased xenograft tumor growth and invasion in mice but resulted in little liquefactive necrosis in tumors. However, GW4869 treatment significantly inhibited xenograft tumor growth but resulted in more liquefactive necrosis. SCC7-derived EVs decreased the expression level of PTPN2, suppressing the immune responses of CD8 + T cells in vivo. Moreover, SCC7-EV treatment significantly enhanced the tumor expression levels of crucial molecules in the IL-17A pathway, including IL-17A, TRAF6 and c-FOS, whereas GW4869 treatment significantly reduced those levels in tumor tissues. CONCLUSION: Our results indicated that OSCC-derived EVs can promote tumor progression by altering the TME, causing an inflammatory cytokine imbalance, inducing immunosuppression, and contributing to overactivation of the IL-17A-induced signaling pathway. Our study might provide novel insights into the role of OSCC-derived EVs in tumor biological behavior and immune dysregulation.

The Promotional Effect of GW4869 on C. albicans Invasion and Cellular Damage in a Murine Model of Oral Candidiasis.

Candida albicans (C. albicans) is one of the most common fungi in the human body; it is an opportunistic pathogen and can cause candidiasis. Extracellular vesicles (EVs) derived from the host cells have a potentially protective effect against pathogens and can be developed as vaccine formulations. GW4869 can inhibit the production and release of EVs. Previous studies have indicated that GW4869 can alter the immune and inflammatory responses of the host. However, the effect of GW4869 on Candida infection and the anti-Candida response of the host has not been investigated. We evaluated the effect of GW4869 on C. albicans invasion, biofilm formation, and cellular damage in a murine model of oral candidiasis. In this study, C. albicans-infected mice were injected with or without GW4869. The results proven by macroscopic, microscopic, and ultramicroscopic methods showed that GW4869 treatment exacerbated the oral candidiasis of mice, promoted C. albicans invasion and biofilm formation, and aggravated oral mucosal inflammation and cellular ultrastructural damage. The results are beneficial in the further exploration of the immune mechanism of C. albicans infection.

Cigarette smoking induces the activation of RIP2/caspase-12/NF-κB axis in oral squamous cell carcinoma.

Cigarette smoking is one of the major risk factors for the occurrence and progression of oral squamous cell carcinoma (OSCC). Receptor-interacting protein 2 (RIP2) has been involved in mucosal immunity and homeostasis via a positive regulation of nuclear factor κB (NF-κB) transcription factor activity. Caspase-12 can bind to RIP2 and dampen mucosal immunity. However, the roles of RIP2/NF-κB and caspase-12 in OSCC induced by cigarette smoking remain unknown. Herein, we investigated the effects of cigarette smoking on the RIP2/NF-κB and caspase-12 in human OSCC tissues and OSCC cell lines (HSC-3). We first observed that RIP2 mediated NF-κB activation and caspase-12 upregulation in OSCC patients with cigarette smoking and cigarette smoke extract (CSE)-treated HSC-3 cells, respectively. Moreover, we confirmed that the downregulation of RIP2 by siRNA resulted in the reduction of caspase-12 expression and NF-κB activity in the presence of CSE treatment in vitro. In summary, our results indicated that cigarette smoking induced the activation of the RIP2/caspase-12/NF-κB axis and it played an important role in the development of OSCC. The RIP2/caspase-12/NF-κB axis could be a target for OSCC prevention and treatment in the future.

A metagenome-wide association study of the gut microbiota in recurrent aphthous ulcer and regulation by thalidomide.

Recurrent aphthous ulcer (RAU), one of the most common diseases in humans, has an unknown etiology and is difficult to treat. Thalidomide is an important immunomodulatory and antitumor drug and its effects on the gut microbiota still remain unclear. We conducted a metagenomic sequencing study of fecal samples from a cohort of individuals with RAU, performed biochemical assays of cytokines, immunoglobulins and antimicrobial peptides in serum and saliva, and investigated the regulation effects of thalidomide administration and withdrawal. Meanwhile we constructed the corresponding prediction models. Our metagenome-wide association results indicated that gut dysbacteriosis, microbial dysfunction and immune imbalance occurred in RAU patients. Thalidomide regulated gut dysbacteriosis in a species-specific manner and had different sustainable effects on various probiotics and pathogens. A previously unknown association between gut microbiota alterations and RAU was found, and the specific roles of thalidomide in modulating the gut microbiota and immunity were determined, suggesting that RAU may be affected by targeting gut dysbacteriosis and modifying immune imbalance. In-depth insights into sophisticated networks consisting of the gut microbiota and host cells may lead to the development of emerging treatments, including prebiotics, probiotics, synbiotics, and postbiotics.

Oral Bacteriome and Mycobiome across Stages of Oral Carcinogenesis.

Oral microbial dysbiosis contributes to the development of oral squamous cell carcinoma (OSCC). Numerous studies have focused on variations in the oral bacterial microbiota of patients with OSCC. However, similar studies on fungal microbiota, another integral component of the oral microbiota, are scarce. Moreover, there is an evidence gap regarding the role that microecosystems play in different niches of the oral cavity at different stages of oral carcinogenesis. Here, we catalogued the microbial communities in the human oral cavity by profiling saliva, gingival plaque, and mucosal samples at different stages of oral carcinogenesis. We analyzed the oral bacteriome and mycobiome along the health-premalignancy-carcinoma sequence. Some species, including Prevotella intermedia, Porphyromonas endodontalis, Acremonium exuviarum, and Aspergillus fumigatus, were enriched, whereas others, such as Streptococcus salivarius subsp. salivarius, Scapharca broughtonii, Mortierella echinula, and Morchella septimelata, were depleted in OSCC. These findings suggest that an array of signature species, including bacteria and fungi, are closely associated with oral carcinogenesis. OSCC-associated diversity differences, species distinction, and functional alterations were most remarkable in mucosal samples, not in gingival plaque or saliva samples, suggesting an urgent need to define oral carcinogenesis-associated microbial dysbiosis based on the spatial microbiome. IMPORTANCE Abundant oral microorganisms constitute a complex microecosystem within the oral environment of the host, which plays a critical role in the adjustment of various physiological and pathological states of the oral cavity. In this study, we demonstrated that variations in the "core microbiome" may be used to predict carcinogenesis. In addition, sample data collected from multiple oral sites along the health-premalignancy-carcinoma sequence increase our understanding of the microecosystems of different oral niches and their specific changes during oral carcinogenesis. This work provides insight into the roles of bacteria and fungi in OSCC and may contribute to the development of early diagnostic assays and novel treatments.

Enzymes and microorganisms jointly promote the fermentation of rapeseed cake.

Rapeseed cake is a by-product of rapeseed oil separation. The nutritional components of rapeseed cake mainly include a variety of carbohydrates, proteins, and minerals. In order to improve the conversion rate of rapeseed cake, we studied the physicochemical properties, the structure of microbial communities, and the composition of metabolites in rapeseed cake after enzymatic fermentation. The results showed that the addition of enzymatic preparation increased microbial diversity. The relative abundance of Bacillus, Lysinibacillus, Empedobacter, Debaryomyces, Hyphopichia, and Komagataella in enzymatic fermentation was significantly higher than that in natural fermentation. Unlike natural fermentation, microbial diversity during enzymatic fermentation is specific, which improves the efficiency of fermentation. Otherwise, enzymatic fermentation promotes the conversion of macromolecular substances in rapeseed cake, which increases small metabolites, such as fatty acids, organic acids, amino acids and their derivatives. The metabolite enrichment pathway is mostly concentrated in sugar metabolism and fatty acid metabolism. In conclusion, after adding enzymatic preparation, enzymes and microorganisms jointly promote the transformation of macromolecules during the fermentation of rapeseed cake, which laid a good foundation for further utilization of rapeseed cake.

Efficacy and safety of miconazole muco-adhesive tablet versus itraconazole in oropharyngeal candidiasis: A randomized, multi-centered, double-blind, phase 3 trial.

Oropharyngeal candidiasis (OPC) is an opportunistic infection treated with anti-fungal agents. Herein, we evaluate the efficacy and safety of miconazole buccal tablets (MBT) and itraconazole capsules in the localized treatment of patients with OPC. In this multi-centered, double-blinded, phase III trial (CTR20130414), both males and non-pregnant females (≥18 years) with OPC were randomized (1:1) to MBT plus placebo (experimental group) or itraconazole capsules plus placebo (control group). The primary endpoint was clinical cure at the end-of-treatment period [visit 4 (V4)] while secondary endpoints were clinical remission rates, partial remission rates, mycological cure, clinical relapse, and adverse events (AEs). All endpoints were statistically analyzed in both the full analysis set (FAS) and per-protocol (PP) set. A total of 431 (experimental: 216; control: 215) subjects were included. At V4, in the FAS set, the clinical cure was achieved in 68% and 59% patients in experimental and control groups, respectively with a treatment difference of 9% [95% confidence interval (CI): -1,19; P < .001] demonstrating non-inferiority of MBT over itraconazole. At V4, mycological cure rates were 68.2% and 42.0% in the experimental group and control groups (P < .001), respectively in FAS. The relapse rates were 5.4% and 6.6%, respectively, in the experimental and control groups. A total of 210 patients experienced AEs during treatment with 47.7% in the experimental group and 49.8% in the control group with no deaths. This study demonstrated that once-daily treatment with MBT was non-inferior to itraconazole with higher mycological cure rates and was tolerable with mild AE in patients with OPC.

Miconazole is an antifungal drug against certain types of fungus or yeast infections. In this study, we showed that treatment with once-daily miconazole buccal tablets was as effective as systemic itraconazole capsules in Chinese patients infected by oropharyngeal candidiasis with minimum side effects.

ZNF667 facilitates angiogenesis after myocardial ischemia through transcriptional regulation of VASH1 and Wnt signaling pathway.

Zinc finger protein 667 (ZNF667, also referred as Mipu1), a widely expressed KRAB/C2H2­type zinc finger transcription factor, can protect against hypoxic­ischemic myocardial injury. Pro­angiogenesis is regarded as a promising strategy for the treatment of acute myocardial infarction (AMI). However, whether ZNF667 is involved in the angiogenesis following AMI remains to be elucidated. The present study reported that the expression of ZNF667 in CD31­positive endothelial cells (ECs) was upregulated in the heart of AMI mice. Hypoxic challenge (1% oxygen) promoted the mRNA and protein expression of ZNF667 in the human umbilical vein endothelial cells (HUVECs) in a time­dependent manner. Moreover, ZNF667 promoted hypoxia­induced invasion and tube formation of HUVECs. Mechanically, ZNF667 could directly bind to the promoter of anti­angiogenic gene VASH1 and inhibit its expression. Consequently, VASH1 overexpression abolished hypoxic challenge or ZNF667 overexpression­induced invasion and tube formation of HUVECs. Further bioinformatic analyses suggested that overexpression of ZNF667 or knockdown of VASH1­induced differentially expressed genes in HUVECs were greatly enriched in the Wnt signaling pathway (DAAM1, LEF1, RAC2, FRAT1, NFATc2 and WNT5A). Together, these data suggested that ZNF667 facilitates myocardial ischemia­driven angiogenesis through transcriptional repression of VASH1 and regulation of Wnt signaling pathway.

Antimicrobial Effect of Extracellular Vesicles Derived From Human Oral Mucosal Epithelial Cells on Candida albicans.

Candida albicans (C. albicans) is a commensal microorganism that colonizes the mucosal surfaces of healthy individuals. Changes in the host or environment can lead to overgrowth of C. albicans and infection of the host. Extracellular vesicles (EVs) are released by almost all cell types and play an increasingly recognized role in fighting microbial infection. The aim of the present study was to assess whether EVs derived from human oral mucosal epithelial (Leuk-1) cells can suppress the growth and invasion of C. albicans. The in vitro efficacy of Leuk-1-EVs against C. albicans was assessed by optical microscopy, laser scanning confocal microscopy, scanning electron microscopy, and transmission electron microscopy. The germ tube formation rate, the percentage of hyphae and the microcolony optical density were also used to analyze the growth of C. albicans in a coculture model with Leuk-1 cells and EVs or after inhibition of the secretion of EVs. A mouse model of oral candidiasis was established and submucosal injection of Leuk-1-EVs in the tongue was performed. Macroscopic observation, H&E staining, PAS staining, and scanning electron microscopy were used to assess antifungal effects of Leuk-1-EVs in vivo. The in vitro results showed that the growth of C. albicans was inhibited and that the morphology and ultrastructure were changed following Leuk-1-EVs treatment. The in vivo results exhibited that white lesions of the tongue, C. albicans infection, and oral mucosal inflammation of the infected mice were significantly alleviated after Leuk-1-EVs treatment. We thus reveal an antifungal capability of EVs derived from oral epithelial cells against C. albicans that is mediated by direct damage effects and potential synergy between EVs and human oral mucosal epithelial cells. This finding offers an intriguing, previously overlooked method of antifungal defense against C. albicans.

Difficult and complicated oral ulceration: an expert consensus guideline for diagnosis.

The complexity of oral ulcerations poses considerable diagnostic and therapeutic challenges to oral specialists. The expert consensus was conducted to summarize the diagnostic work-up for difficult and complicated oral ulcers, based on factors such as detailed clinical medical history inquiry, histopathological examination, and ulceration-related systemic diseases screening. Not only it can provide a standardized procedure of oral ulceration, but also it can improve the diagnostic efficiency, in order to avoid misdiagnosis and missed diagnosis.

Altered protein profile of plasma extracellular vesicles in oral squamous cell carcinoma development.

Extracellular vesicles (EVs) are involved in a wide range of pathological processes and recognized as potential and novel biomarkers for oral squamous cell carcinoma (OSCC). Here, we describe the plasma EV proteome of rats with 4-nitroquinoline-1-oxide (4NQO)-induced OSCC or moderate dysplasia (MD), which can progress to OSCC, by tandem mass tag (TMT)-labeled mass spectrometry. The proteomic profiles suggest the differential expression of various proteins in MD and OSCC, some well-recognized pathological changes (e.g., translation, ATP metabolism, and mesenchymal transition), and some novel pathological changes (e.g., podosome, focal adhesion, and S100 binding). We re-examined the presence of traditional exosomal markers and the reported novel pan-EV markers. In summary, these results suggest potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers. SIGNIFICANCE: This research suggests potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers.

Active Smoking Induces Aberrations in Digestive Tract Microbiota of Rats.

Cigarette smoking could have certain effects on gut microbiota. Some pioneering studies have investigated effects of active smoking on the microbiome in local segments of the digestive tract, while active smoking-induced microbiome alterations in the whole digestive tract have not been fully investigated. Here, we developed a rat model of active smoking and characterized the effects of active smoking on the microbiota within multiple regions along the digestive tract. Blood glucose and some metabolic factors levels, the microbial diversity and composition, relative abundances of taxa, bacterial network correlations and predictive functional profiles were compared between the control group and active smoking group. We found that active smoking induced hyperglycemia and significant reductions in serum insulin and leptin levels. Active smoking induced region-specific shifts in microbiota structure, composition, network correlation and metabolism function along the digestive tract. Our results demonstrated that active smoking resulted in a reduced abundance of some potentially beneficial genera (i.e. Clostridium, Turicibacter) and increased abundance of potentially harmful genera (i.e. Desulfovibrio, Bilophila). Functional prediction suggested that amino acid, lipid, propanoate metabolism function could be impaired and antioxidant activity may be triggered. Active smoking may be an overlooked risk to health through its potential effects on the digestive tract microbiota, which is involved in the cause and severity of an array of chronic diseases.

Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and M2 polarization of macrophage.

Oral immunosuppression caused by smoking creates a microenvironment to promote the occurrence and development of oral mucosa precancerous lesions. This study aimed to investigate the role of metabolism and macrophage polarization in cigarette-promoting oral leukoplakia. The effects of cigarette smoke extract (CSE) on macrophage polarization and metabolism were studied in vivo and in vitro. The polarity of macrophages was detected by flow cytometric analysis and qPCR. Liquid chromatography-mass spectrometry (LC-MS) was used to perform a metabolomic analysis of Raw cells stimulated with CSE. Immunofluorescence and flow cytometry were used to detect the polarity of macrophages in the condition of glutamine abundance and deficiency. Cell Counting Kit-8 (CCK-8), wound-healing assay, and Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) double-staining flow cytometry were applied to detect the growth and transferability and apoptosis of Leuk-1 cells in the supernatant of Raw cells which were stimulated with CSE, glutamine abundance and deficiency. Hyperkeratosis and dysplasia of the epithelium were evident in smoking mice. M2 macrophages increased under CSE stimulation in vivo and in vitro. In total, 162 types of metabolites were detected in the CSE group. The metabolites of nicotine, glutamate, arachidic acid, and arginine changed significantly. The significant enrichment pathways were also selected, including nicotine addiction, glutamine and glutamate metabolism, and arginine biosynthesis. The results also showed that the supernatant of Raw cells stimulated by CSE could induce excessive proliferation of Leuk-1 and inhibit apoptosis. Glutamine abundance can facilitate this process. Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and macrophage M2 polarization.

[Characteristics and Sources of VOCs at Different Ozone Concentration Levels in Tianjin].

To further study the effect of volatile organic compounds (VOCs) on ozone pollution, the characteristics and sources of VOCs at different ozone (O3) concentration levels were analyzed, using high-resolution online monitoring data obtained from Tianjin in the summer of 2019. Results showed that VOCs concentrations were 32.94, 38.10, 42.41, and 47.12 µg ·m-3, when the O3 concentration levels were categorized as excellent, good, light pollution, and moderate pollution, respectively. VOCs were composed of alkanes, alkenes, alkynes and aromatics, which accounted for 61.72%-63.36%, 14.96%-15.51%, 2.73%-4.13%, and 18.53%-19.10%, respectively, of VOCs concentrations at different O3 concentration levels. Among them, the proportion of alkanes was slightly higher when O3 concentration was categorized as good or light pollution, alkenes and alkynes accounted for the highest proportion when O3 concentration was excellent, and the proportion of aromatics was highest during periods of moderate pollution. The main VOCs species were propane, ethane, ethylene, toluent, n-butane, isopentane, m/p-xylene, propylene, acetylene, n-hexane, isobutene, benzene, n-pentane, isoprene, and 1,2,3-trimethylbenzene. The concentration percentage of isopentane, n-pentane, benzene, ethylene, propylene, n-butane, and isobutane increased gradually as O3 concentration increased. Significant increases in isoprene and 1,2,3-trimethylbenzene were observed during periods of light and moderate pollution. Alkenes and aromatics had higher ozone formation potential (OFP), and the contribution of alkenes to OFP declined as the O3 level rose, whereas that of aromatics increased. Ethylene, propylene, m/p-xylene, 1,2,3-trimethylbenzene, toluene, isoprene, trans-2-butene, and cis-2-pentene were the key species for O3 generation, and the contribution ratio of 1,2,3-trimethylbenzene, isoprene, propylene, and ethylene to OFP increased significantly during light or moderate O3 pollution. Positive matrix factorization was applied to estimate the source contributions of VOCs. Automobile exhaust, solvent usage, liquefied petroleum gas (LPG)/gasoline evaporation, combustion, petrochemical industrial emissions, natural sources, and other industrial emissions were identified as major sources of VOCs in summer. As O3 concentration level rose, the contribution percentage of automobile exhaust, LPG/gasoline evaporation, petrochemical industrial emissions, and natural sources increased gradually, whereas the contribution of combustion and other industrial emissions decreased overall. The contribution of solvent usage was lower when O3 levels indicated light or moderate pollution than when it was good.

Smoking increases oral mucosa susceptibility to Candida albicans infection via the Nrf2 pathway: In vitro and animal studies.

Smoking and Candida albicans (C. albicans) infection are risk factors for many oral diseases. Several studies have reported a close relationship between smoking and the occurrence of C. albicans infection. However, the exact underlying mechanism of this relationship remains unclear. We established a rat infection model and a C. albicans-Leuk1 epithelial cell co-culture model with and without smoke exposure to investigate the mechanism by which smoking contributes to C. albicans infection. Oral mucosa samples from healthy individuals and patients with oral leucoplakia were also analysed according to their smoking status. Our results indicated that smoking induced oxidative stress and redox dysfunction in the oral mucosa. Smoking-induced Nrf2 negatively regulated the NLRP3 inflammasome, impaired the oral mucosal defence response and increased the oral mucosa susceptibility to C. albicans. The results suggest that the Nrf2 pathway could be involved in the pathogenesis of oral diseases by mediating an antioxidative response to cigarette smoke exposure and suppressing host immunity against C. albicans.