[Serum metabonomics study on Cr (â ¥ ) subchronic exposure rats based on UPLC-Q-TOF-MS/MS platform].

Objective: To ananlyze the toxic effects and mechanisms of Cr (Ⅵ) subchronic exposure based on metabonomics techniques. Methods: Twenty-nine female Sprague-Dawley rats were randomly divided into control group, low dose group and high dose group, 10, 9, 10, respectively. The control group, low dose group and high dose group were treated with 0, 10, 50 mg/L Cr (Ⅵ) for 90 days respec tively. The serum samples of rats with different dose of Cr (Ⅵ) treatment were detected Using UPLC-Q-TOF-MS/MS technique and data was analyzed by PCA, PLS-DA and OPLS-DA to compare with metabolic profile in different Cr (Ⅵ) dose treatments. Pathway analysis was performed using MetaboAnalyst 4.0 software. Results: UPLC-Q- TOF-MS/MS has stable detection performance and reliable experimental data. The control group, low Cr (Ⅵ) and high Cr (Ⅵ ) metabolic profiles of rats serum differences was obviously, and there is significant difference of serum metabolic profile among rats treated with different dose of Cr (Ⅵ) . 18 differential metabolites were screened between Cr (Ⅵ) low dose group and control group, 23 differential metabolites between Cr (Ⅵ) high dose group and control group. Compared to control group, there were 13 differential metabolites in both Cr (Ⅵ) high dose group and Cr (Ⅵ ) low dose group, such as 3-Hydroxy-11Z-octadecenoylcarnitine, Anserine, Farnesyl pyrophosphate, Linoleoyl ethanolamid e, Linoleyl carnitine, Lithocholate 3-O-glucuronide, LysoPC [20∶2(11Z, 14Z) ], LysoPC[20∶3 (5Z, 8Z, 11Z) ], LysoPC[22∶2(13Z, 16Z) ], PG[16∶0/22∶5(7Z, 10Z, 13Z, 16Z, 19Z) ], PI[18∶1 (11Z) /20∶4(5Z, 8Z, 11Z, 14Z) ], PI[20∶3(5Z, 8Z, 11Z) /18∶0], Serotonin. These differential metabolites were related to Glycerophospholipid metabolism, Tryptophan metabolism, Pentose and glucuronate interconversions, Terpenoid backbone biosynthesis. Conclusion: Cr (Ⅵ) subchronic exposure could induce the significant difference of serum metabolic profile. The differential metabolites induced by Cr (Ⅵ) subchronic ex- posure were mainly related to amino acid and lipid metabolism.

[Effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes in the murine model of vaginal candidiasis].

OBJECTIVE: To study the effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes on candida albicans growth. METHODS: (1)Thirty female Kunming mice were treated with estrogen and white candida yeast suspension were poured into vagina to establish a vulvovaginal candidiasis(VVC)murine model. After successful establishing the VVC mouse model, mice were randomly sorted into test group(n=15)and control group(n=15). Suspension(30 µl, 100 µg/ml)of recombinant peptide LL-37 expressed and purified in Prokaryotes was given by intravaginal administration to the test group for 5 days, while the same amount of phosphate buffered saline(PBS)was given to the control group.(2)Tweenty-four hours after treatment, the fungal burden and colony-forming unit(CFU)of vaginal fluids were evaluated. All mice were subsequently sacrificed and vaginal tissues were harvested for tissue homogenate preparation. ELISA was used to determine the levels of nterleukin-10(IL-10)and interferon-γ(IFN-γ)in the isolated vaginal tissues. RESULTS: (1)VVC mouse model was established successfully in this study. Vaginal mucosa congestion, edema, vaginal plica disappearing were obviously observed in the control group. After treatment with recombinant protein LL-37 vaginal mucosa has no obvious change in the test group.(2)Fungal burden and CFU of vaginal fluids were significantly lower in the test group [(4.8±1.0)×10(4) CFU/ml]than that in the control group [(8.5±2.1)×10(4) CFU/ml, P=0.017]. IFN-γ level of the test group was increased [(257±11)vs(197±4)pg/ml, P=0.000], while the level of IL-10 was reduced [(287 ± 15)vs(379 ± 17)pg/ml P=0.000] resulting in a the ratio of IFN-γ/IL-10 was in significantly higher in test group(0.892±0.008 vs 0.496±0.013, P=0.000). CONCLUSION: Recombinant protein LL-37 expressed and purified from prokaryotes inhibits the growth candida albicans and improves vaginal immunity by adjusting IFN-γ and IL-10 secretion in the VVC mouse model, highlighting the therapeutic potential of LL-37 for VVC.

[Prokaryotic expression and purification of antimicrobial peptide LL-37 and the inhibiting effect against Candida albicans].

OBJECTIVE: To study the inhibitory effect of antimicrobial peptide LL-37 on Candida albicans through its ability to promote the secretion of immune factors by vaginal epithelial cells. METHODS: (1) LL-37 prokaryotic expression vector pET-Duet/LL-37 was constructed and its expression was induced in Escherichia coli M15. The expressed LL-37 fusion protein was purified and identified by western blot. Antifungal activity of the purified protein was initially identified by Kirby-Bauer (K-B) method. (2) Purified LL-37 protein was added to human vaginal epithelial cells co-cultured with Candida, and inhibitory effect on Candida growth was determined by the glucose consumption method. Interferon γ (IFN-γ), interleukin 10 (IL-10) concentration and IFN-γ/IL-10 ratio were measured by ELISA at different time points. RESULTS: (1) LL-37 fusion protein was purified to 96% purity at a concentration of 433.92 µg/ml, and was shown to possess anti-fungal activity confirmed by the K-B method. (2) A Candida-vaginal epithelial cells co-culture system was successfully constructed. LL-37 recombinant protein inhibited the growth of Candida with absorbance values significantly higher in the treatment group compared to the control group at all measured time points (12-hour: 3.008±0.003 versus 2.967±0.003, 24-hour: 2.941±0.003 versus 2.601±0.003, 48-hour: 2.893 ± 0.004 versus 2.409 ± 0.003; all P<0.01). Furthermore, the rate of decrease was also much slower compared to the control group. In both control and experimental groups, IFN-γ and IL-10 secretion levels were observed to rise at first peaking at 24 hours and subsequently decrease. For each time period, IFN-γ concentration in the experimental group was significantly higher at 24 hours compared to the control group [(104.00 ± 1.07) versus (85.17 ± 0.28) pg/ml,P<0.01]. In contrast, IL-10 concentrations were significantly lower than the control group at all time points (P<0.01). IFN-γ/IL-10 ratio was also observed to be significantly higher than the control group at all measured time points (P<0.01). CONCLUSIONS: (1) Recombinant protein LL-37 could significantly inhibit the growth of Candida. (2) By influencing the secretion of immune factors such as IFN-γ, IL-10, etc, recombinant protein LL-37 is able to adjust vaginal epithelial cells local immunity, and enhance resistance to Candida infection.

Wrist ROM measurements using smartphone photography: Reliability and validity.

No one has validated measuring the wrist's active range of motion (ROM) using smartphone images in patients. It is not known whether pathological factors affect the accuracy of this measurement technique. The purpose of this study was to assess if smartphone photography is as reliable and valid as manual goniometry for measuring wrist joint ROM. We reviewed 38 wrists in 38 patients (21 women and 17 men) with a mean age of 45 years (range, 26-60). Smartphones were used to take digital photos of injured wrists at extremes of wrist motion, including flexion, extension, radial and ulnar deviation. The mean difference in measured ROM between the two measurement methods (digital photos and handheld goniometer) was compared using Student's t test and the relationship determined using Pearson correlation coefficients. Bland-Altman analysis was used to define the limits of agreement (LOA). No significant difference was found when comparing the wrist ROM in the four positions using manual goniometry and digital measurements from photos taken by a surgeon. Between the goniometer measurements and digital photos taken by a surgeon, the Pearson coefficients were high, with most being above 0.8 for the four positions. The Pearson coefficients also show the smartphone measurements were highly precise. There was high reliability between the photographs taken by surgeons and by patients, as well as high interobserver reliability. Smartphone photography is a reliable and valid method to measure wrist joint ROM in patients. This measurement method can be used to measure outcomes.

Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100.

To study the functions of the 13 gvp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gvp gene cluster and introduced a kanamycin resistance (kappa) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gvp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the kappa cassette in any of the gvp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the kappa cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the kappa insertions resulted in phenotypic reversion. Electron microscopic analysis of the kappa mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicle than in the wild type, while interruptions of gvpF, -G, -H, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate the gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL, -K, -J, -I, -H, -G, and -F, which have the leftward transcriptional orientation encode proteins involved in early steps in the assembly of gas vesicles.