A comparison of size exclusion chromatography-based tandem strategies for plasma exosome enrichment and proteomic analysis.

Proteomic analysis of exosomes from human plasma faces a tremendous challenge mainly due to the low abundance of the exosome itself and the complexity of the plasma matrix. Therefore, enrichment of exosomes from human plasma is an essential and indispensable step for large scale and in depth proteomic analysis. Size exclusion chromatography (SEC) is one of the most extensively used methods for exosome isolation from human plasma and many SEC-based tandem methods were established in order to increase the purity of the enriched exosomes and thus the accuracy of the proteomic analysis. To compare the advantages and disadvantages of the different isolation methods and subsequently to promote the establishment of a standardized method for plasma proteomic research, the capacities of the direct SEC method, the combination of SEC with ultracentrifugation (SEC-UC), ultrafiltration (SEC-UF), and titanium dioxide microspheres (SEC-TiO2) were systematically evaluated for exosome isolation from human plasma and thus proteomic analysis. The results demonstrated that the SEC-based tandem methods were superior to the direct SEC method in the purity of exosomes isolated from human plasma. Additionally, the SEC-UC method possessed the highest number of the total identified proteins and the overlapped proteins with the top 100 exosome markers in comparison with the other methods. The SEC-TiO2 method displayed the biggest capacity for plasma protein deleting. We expect that the research will have more beneficial values in the field of exosome research.

[Analysis of vaginal microecology in 23 181 cases of the gynecological female outpatients].

Objective: To analyze the vaginal microecological status of vaginitis population and non-vaginitis population of gynecological female outpatients. Methods: A total of 30 265 women who visited the gynecological outpatient clinic of Beijing Obstetrics and Gynecology Hospital from December 2018 to December 2020 completed vaginal microecological examination. After removing the follow-up patients, 23 181 women were divided into group with symptoms and signs of vaginitis (6 697 cases) and group without symptoms and signs of vaginitis (16 484 cases), according to whether the women with symptoms and signs of vaginitis or not. And the vaginal microecological status of the two groups was compared and analyzed. Results: (1) The total detection rate of vaginitis in the initial women was 34.87% (8 083/23 181), of which 46.10% (3 087/6 697) in group with symptoms and signs of vaginitis and 30.31% (4 996/16 484) in group without symptoms and signs of vaginitis, nearly 1/3 of the gynecological outpatients without signs and symptoms of vaginitis had vaginitis. (2) Among the types of simple vaginitis, vulvovaginal candidiasis (VVC) was the most frequent in group with symptoms and signs of vaginitis (16.01%, 1 072/6 697), followed by aerobic vaginitis (AV; 12.83%, 859/6 697), with significant differences compared with group without symptoms and signs of vaginitis (all P<0.001). There were no statistical differences between the two groups of bacterial vaginosis (BV) and trichomonal vaginitis (TV), indicating that BV and TV were more likely to be neglected (all P>0.05). (3) The proportion of various combinations of vaginitis among 2 632 cases of mixed vaginitis were, in descending order: BV+AV, VVC+AV, BV+AV+VVC, AV+TV, AV+TV+BV, BV+VVC. (4) Microecological analysis of 15 098 cases diagnosed with non-vaginitis had normal flora (including those with normal flora and those with normal flora but decreased function) in 14 013 cases (92.81%, 14 013/15 098), abnormal flora in 429 cases (2.84%, 429/15 098) and the BV intermediate in 656 cases (4.34%, 656/15 098); this indicated that the vast majority of the microecological tests were normal in the vaginal microbiota of those without vaginitis. Conclusions: Microecological examination could diagnose multiple pathogenic infections at once, and is especially important as a guide for the definitive diagnosis of mixed vaginitis and vaginitis with atypical clinical symptoms. Vaginal infections such as BV and TV that are easily overlooked should be concerned.

[Dynamics of vaginal microbiota in women of reproductive age during the menstrual cycle].

Objective: To investigate the dynamic changes of vaginal microbiota in different phases of menstrual cycle in healthy Chinese women of childbearing age. Methods: A total of 11 healthy women of childbearing age with regular menstruation, who had physical examination in the Gynecology Clinic of Beijing Obstetrics and Gynecology Hospital from September to December 2020 were randomly selected as research subjects. Vaginal secretions were collected during menstrual phase (2nd-3rd day), mid-follicular phase (7th-8th day), and mid-luteal phase (21st-22nd day) for microbiota analysis through metagenomic sequencing. Results: (1) Vaginal microbiota species were the most diverse in menstrual phase and the least in follicular phase, observing dominant vaginal bacteria gradually changing to Lactobacillus from menstrual phase to follicular phase and then to luteal phase. (2) The dynamic evolution of vaginal microbiota from menstrual phase to follicular phase and then to luteal phase was divided into: no change in dominant bacteria, replacement of dominant bacteria, changes in the proportion of dominant bacteria, and recurrence of dominant bacteria (non-Lactobacillus-dominance appeared again in luteal phase after returning to normal Lactobacillus-dominance in follicular phase). (3) Prevotella, especially Prevotella_bivia, was significantly higher during menstrual phase. Conclusions: Healthy vaginal microbiota should be relatively stable, but also have the ability of dynamic change and self-recovery. Prevotella plays a central role among opportunistic pathogens in the vagina, whose function remains to be investigated.

Identification of peroxiredoxin 6 as a potential lung-adenocarcinoma biomarker for predicting chemotherapy response by proteomic analysis.

The prognosis of lung cancer remains poor due to the limited biomarker selection for treating patients with optimal chemotherapy. The aim of this study is to discover and identify new biomarkers with the value of predicting chemotherapy responses in a lung adenocarcinoma (AD) specimen. In this study, six pairs of pre-treatment fresh primary lung AD-cancer tumors with varied chemotherapy responses were used to discover new biomarkers by two-dimensional difference gel electrophoresis (2D DIGE). Among the matched protein spots, 19 were up-regulated and 18 were down-regulated in chemo-sensitive tumors versus chemo-resistant tissues. These differentially expressed proteins could be divided into five classes: redox regulation protein, the cytoskeletal protein, cell metabolism enzymes or proteins, apoptosis, signal transduction mediated molecules, and other functional proteins. Proteins of interest, including PRDX2, PRDX6, and Gelsolin, were differentially expressed in chemo-sensitive tumors versus chemo-resistant tissues and these observations were validated by immunohistochemistry in 92 formalin-fixed and paraffin-embedded (FFPE) specimens. Our results demonstrated that PRDX6 protein expression was closely related to tumor response (cc2 = 5.57, P < 0.05), whereas no relationship of PRDX2 and Gelsolin were obtained with tumor response (cc2 = 0.51 P > 0.05, cc2 = 0.41 P > 0.05). This tissue proteomics study provides evidence that PRDX6 may be regarded as a predictive biomarker for poor chemotherapy response, which can be helpful in guiding pretreatment protocols.

[Analysis of homology and drug sensitivity of vaginal isolates of 10 patients with recurrent vulvovaginal candidiasis in recurrent episodes].

Objective: To detect karyotype homology of vaginal isolates from patients with recurrent vulvovaginal candidiasis (RVVC) in recurrent episodes, and to discuss changes of susceptibility of Candida strains to antifungal drugs with clinical progress. Method: s Ten patients were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University from September 2018 to June 2019, who were firstly diagnosed with RVVC. Vaginal discharges were collected before first treatment and after first relapse. Vaginal strains were isolated, purificated and identificated. Then karyotype of 20 strains isolated from 10 patients were detected by restriction endonuclease analysis of genomic DNA (REAG) using enzyme BssHⅡand pulsed field gel electrophoresis (PFGE) methods, and sensitivity of clinical isolates to 5 antifungal drugs (clostridium, fluconazole, miconazole, itraconazole and nystatin) was also detected using disk diffusion method. Result: s (1) All 20 strains of 10 patients with RVVC were Candida albicans, and their chromosomes were extremely similar after BssHⅡ enzyme digestion. The gene bands of isolated strains from the same patient were completely identical. (2) After clinical medication, the sensitivity of vaginal isolates to azoles was generally decreased, but remained highly sensitive to nystatin, nystatin (first and second clinical isolates: 100% sensitivity and 100% sensitivity)>clotrimazole (100% sensitivity and 90% sensitivity)>fluconazole (80% sensitivity and 70% sensitivity)>itraconazole (60% sensitivity and 50% sensitivity)>miconazole (30% sensitivity and 20% sensitivity). Conclusions: (1) The latency of the same colonized strain in the vagina may be the cause of repeated RVVC episodes. (2) Antifungal agents could selectively induce drug resistance to Candidas, and Candidas show cross-resistance to antifungal agents. Repeated fungal culture and drug sensitivity test in patients with RVVC are very necessary for correct selection of antifungals.

[Changes of local vaginal immune regulation in rats infected with vulvovaginal candidiasis].

Objective: To study the disease process of vulvovaginal candidiasis (VVC) infection in rat model of VVC, and to study the immuno-repairing effect of different treatments on vaginal epithelium and the ultra-structural changes of vaginal epithelial cells. Methods: The VVC model of female rats were established. After successful modeling, the rats were treated with no treatment (model control group), nystatin and Kangfu Xiaoyan suppository. The vaginal epithelium was observed by transmission electron microscopy and immunohistochemical staining. The ultra-structural changes of epithelial cells and the expression of cytokines interferon γ (IFN-γ), interleukin (IL) 4, IL-17 and IgG in epithelial cells were observed and analyzed statistically. Results: The negative conversion rate of model control group was 0, and that of nystatin group was 6/6, and that of Kangfu Xiaoyan suppository group was 5/6; significant difference existed between nystatin, Kangfu Xiaoyan suppository group and model control group (P<0.05). The ultrastructures of vaginal epithelial cells were damaged obviously after VVC infection, and the ultrastructures were repaired by nystatin and Kangfu Xiaoyan suppository under transmission electron microscope. Immunohistochemical staining showed, the expressions of IFN-γ and IgG in the four cytokines which played a protective role increased after Kangfu Xiaoyan suppository treatment, significantly different from that of model control group (P<0.05), but there were no significant differences of the IFN-γ and IgG expression between Kangfu Xiaoyan suppository group and those of nystatin group (P>0.05); the expression of IL-17 was increased after nystatin treatment, but decreased after Kangfu Xiaoyan suppository treatment, and the difference between the two groups had statistical significance (P<0.05). Conclusions: The ultrastructure of vaginal epithelial cells after VVC infection could be damaged obviously, the local immune state is disordered, and the antifungal drug nystatin has a good therapeutic effect on VVC, it could significantly repair the damaged vaginal epithelium structure after VVC infection and strengthen the protective immune function of vaginal epithelium. Kangfu Xiaoyan suppository, one of Chinese medicine, has similar therapeutic effect with nystatin.

[Primary study on the relationship between high-risk HPV infection and vaginal cervical microbiota].

Objective: To understand characteristics of vaginal cervical microbiota in high-risk HPV (hrHPV) infected women and to uncover the relationship between hrHPV infection and vaginal cervical microbiota. Methods: All participants were randomly selected from Peking University First Hospital from September to October of 2017, including 5 subjects of control group, 5 cases of HPV16/18 group, 5 cases of other hrHPV infected group and 3 cases of cervical squamous carcinoma group. All subjects were required to fill in a questionnaire, and cervical and vaginal discharges were separately collected for microscopic examination and new generation sequencing targeting the variable region (V3-V4) of bacterial 16S rRNA gene. Results: Vaginal microbiota analysis: (1) 6 major phylum were found in vaginal microbiota:Firmicutes, Bacteroidetes, Fusobacteria, Actinobacteria, Tenericutes and Proteobacteria. Firmicutes contributed to the majority of normal vaginal flora, Bacteroidetes and Fusobacteria increased in hrHPV infected ones, while Fusobacteria showed significant difference in cervical carcinoma group. (2) Lactobacillus occupied most of normal vaginal flora while genus like Gardnella, Prevotella, Atopobium, Megasphaera and Sneathia increased in hrHPV infected subjects, Sneathia showed significant difference in cervical carcinoma group. (3) No significant difference had been calculated in Alpha diversity of four groups (P=0.073) . Cervical microbiota analysis: (1) Microbial diversity of cervical microbiota was higher than that of vaginal microbiota. (2) Significant difference had been found in Alpha diversity of four groups (P=0.046) . (3) Proteobacteria in normal cervical flora was much more than that in vagina, and Proteobacteria increased significantly in hrHPV infected cervical discharge. (3) Chlamydia increased significantly in cervical carcinoma group. Conclusions: The diversity of cervical microbiota is higher than that of vaginal microbiota. Change in cervical microbiota is more obvious than that of vagina in hrHPV infected subjects. Fusobacteria-Sneathia and Chlamydia significantly increase in cervical carcinoma group. Proteobacteria might relate to hrHPV infection.