PgtE Enzyme of Salmonella enterica Shares the Similar Biological Roles to Plasminogen Activator (Pla) in Interacting With DEC-205 (CD205), and Enhancing Host Dissemination and Infectivity by Yersinia pestis.

Yersinia pestis, the cause of plague, is a newly evolved Gram-negative bacterium. Through the acquisition of the plasminogen activator (Pla), Y. pestis gained the means to rapidly disseminate throughout its mammalian hosts. It was suggested that Y. pestis utilizes Pla to interact with the DEC-205 (CD205) receptor on antigen-presenting cells (APCs) to initiate host dissemination and infection. However, the evolutionary origin of Pla has not been fully elucidated. The PgtE enzyme of Salmonella enterica, involved in host dissemination, shows sequence similarity with the Y. pestis Pla. In this study, we demonstrated that both Escherichia coli K-12 and Y. pestis bacteria expressing the PgtE-protein were able to interact with primary alveolar macrophages and DEC-205-transfected CHO cells. The interaction between PgtE-expressing bacteria and DEC-205-expressing transfectants could be inhibited by the application of an anti-DEC-205 antibody. Moreover, PgtE-expressing Y. pestis partially re-gained the ability to promote host dissemination and infection. In conclusion, the DEC-205-PgtE interaction plays a role in promoting the dissemination and infection of Y. pestis, suggesting that Pla and the PgtE of S. enterica might share a common evolutionary origin.

Iodometric spectrophotometric determination of peroxydisulfate in hydroxylamine-involved AOPs: 15 min or 15 s for oxidative coloration?

In this study, iodometric spectrophotometry, the most-used method for detecting peroxydisulfate (PDS), was modified by increasing the concentration of potassium iodide (KI) for realizing the immediate PDS determination and avoiding the interference of hydroxylamine. Kinetic studies showed that the reaction between PDS and I- to generate the yellow-colored I3- followed the kinetic equation as [Formula: see text] . Detection time of the iodometric spectrophotometry was shortened from 15 min to 15 s when KI concentration was increased from 0.6 M to 4.8 M. Different with the previous iodometric spectrophotometry, the modified method using 4.8 M KI as the indicator was well tolerable to the interference of hydroxylamine at acidic pH conditions. The calibration curve of the modified method showed a well linear relationship (R2 = 0.999) between the absorbance of I3- at 352 nm and PDS concentration in the range of 0-80 µM. The modified method was highly sensitive with the absorptivity of 2.5 × 104 M-1 cm-1 and the limit of detection of 0.11 µM. Moreover, the modified method was successfully applied for monitoring the change of PDS concentration during the degradation of diclofenac with four different PDS-based AOPs, the calculated reaction stoichiometric efficiency (RSE(%)=DiclofenacdegradedPDSconsumed×100%) followed the order as heat/PDS system > hydroxylamine/Fe2+/PDS system > hydroxylamine/Cu2+/PDS system > Fe2+/PDS system.

ABTS as an electron shuttle to accelerate the degradation of diclofenac with horseradish peroxidase-catalyzed hydrogen peroxide oxidation.

Horseradish peroxidase (HRP)-catalyzed hydrogen peroxide (H2O2) oxidation could degrade a variety of organic pollutants, but the intrinsic drawback of slow degradation rate limited its widespread application. In this study, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was introduced into HRP/H2O2 system as an electron shuttle to enhance diclofenac degradation under neutral pH conditions. The green-colored ABTS radical (ABTS•+), generated by the oxidation of ABTS with HRP-catalyzed H2O2 oxidation, was proved to be the main reactive species for the rapid degradation of diclofenac in HRP/H2O2/ABTS system. There was no destruction of ABTS/ABTS•+ in HRP/H2O2/ABTS system, and ABTS was verified as an ideal electron shuttle. The reaction conditions including solution pH (4.5-10.5), HRP concentration (0-8 units mL-1) and H2O2 concentration (0-500 µM) would impact the formation of ABTS•+, and affect the degradation of diclofenac in HRP/H2O2/ABTS system. Moreover, compared with Fenton and hydroxylamine/Fenton systems, HRP/H2O2/ABTS system had better diclofenac degradation efficiency, higher H2O2 utilization efficiency and stronger anti-interference capacity in actual waters. Overall, the present study provided a meaningful and promising way to enhance the degradation of organic pollutants in water with HRP-catalyzed H2O2 oxidation.

Distinct Patterns of Host Adherence by Neisseria gonorrhoeae Isolated from Experimental Gonorrhea.

Neisseria gonorrhoeae (N. gonorrhoeae, gonococci, or GC), the etiologic agent of gonorrhea, is a human-obligate bacterial pathogen. The GC surface contains pili that mediate the adherence to host cells. Studies have shown that GC pili, coded by pilin genes, undergo remarkable changes during human experimental gonorrhea, possibly generated by DNA phase variation during infection. The question that arises is whether the changes in pilins can alter the adherence capacity of N. gonorrhoeae to host cells. In this study, six variants initially isolated from male volunteers infected with one single clone of GC were examined for their adherence patterns with human Chang conjunctiva cells. In this study, we showed that the variants showed distinct adherence patterns to this cell line under light microscopy and scanning electron microscopy. Moreover, two reisolates showed higher adherence capacities than that of the input strain. The results provide an additional example as to how the pilus variation may play a role in the pathogenesis of N. gonorrhoeae.

Progesterone Suppresses Neisseria gonorrhoeae-Induced Inflammation Through Inhibition of NLRP3 Inflammasome Pathway in THP-1 Cells and Murine Models.

Asymptomatic/subclinical gonococcal infections in females continue to be prevalent within the general population, thus emerging as a global health problem. However, the reasons for these clinical manifestations are unknown. Our group had previously found out that in females, asymptomatic gonococcal infections correlate with higher serum progesterone (P4) levels and lower IL-1ß levels in cervical secretions. We used murine infection model and THP-1 cells to determine whether P4 exerts anti-inflammatory effects on gonococcal infections. In the murine infection model, P4 (1 mg/day) inhibited the inflammatory effects induced by gonococcal infections which led to decreased neutrophil infiltration, reduced polymorphonuclear neutrophils (PMNs) numbers, IL-1ß, TNF-α, and IL-6 levels in vaginal secretions. In addition, P4 down-regulated the mRNA and protein levels of NLRP3, associated with lower mRNA levels of pro-IL-1ß, repressed caspase-1 activity in genital tissues and THP-1 cells. Moreover, P4 suppressed the phosphorylation levels of NF-κB and attenuated Neisseria gonorrhoeae (N. gonorrhoeae, gonococci or GC)-induced ROS generation. This is consistent with the two signals required for activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. In conclusion, our result shows that P4 suppresses the gonococci induced-inflammation, especially through the NLRP3 inflammasome pathway, and partially explains the pathogenesis of asymptomatic GC infection in women.

Invasiveness of the Yersinia pestis ail protein contributes to host dissemination in pneumonic and oral plague.

Yersinia pestis, a Gram-negative bacterium, is the etiologic agent of plague. A hallmark of Y. pestis infection is the organism's ability to rapidly disseminate through an animal host. Y. pestis expresses the outer membrane protein, Ail (Attachment invasion locus), which is associated with host invasion and serum resistance. However, whether Ail plays a role in host dissemination remains unclear. In this study, C57BL/6J mice were challenged with a defined Y. pestis strain, KimD27, or an isogenic ail-deleted mutant derived from KimD27 via metacarpal paw pad inoculation, nasal drops, orogastric infection, or tail vein injection to mimic bubonic, pneumonic, oral, or septicemic plague, respectively. Our results showed that ail-deleted Y. pestis KimD27 lost the ability to invade host cells, leading to failed host dissemination in the pneumonic and oral plague models but not in the bubonic or septicemic plague models, which do not require invasiveness. Therefore, this study demonstrated that whether Ail plays a role in Y. pestis pathogenesis depends on the infection route.

Multi-wavelength spectrophotometric determination of hydrogen peroxide in water by oxidative coloration of ABTS via Fenton reaction.

In this study, a sensitive and low-cost multi-wavelength spectrophotometric method for the determination of hydrogen peroxide (H2O2) in water was established. The method was based on the oxidative coloration of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) via Fenton reaction, which resulted in the formation of green radical (ABTS•+) with absorbance at four different wavelengths (i.e., 415 nm, 650 nm, 732 nm, and 820 nm). Under the optimized conditions (CABTS = 2.0 mM, CFe2+ = 1.0 mM, pH = 2.60 ± 0.02, and reaction time (t) = 1 min), the absorbance of the generated ABTS•+ at 415 nm, 650 nm, 732 nm, and 820 nm were well linear with H2O2 concentrations in the range of 0-40 µM (R2 > 0.999) and the sensitivities of the proposed Fenton-ABTS method were calculated as 4.19 × 104 M-1 cm-1,1.73 × 104 M-1 cm-1, 2.18 × 104 M-1 cm-1, and 1.96 × 104 M-1 cm-1, respectively. Meanwhile, the detection limits of the Fenton-ABTS method at 415 nm, 650 nm, 732 nm, and 820 nm were respectively calculated to be 0.18 µM, 0.12 µM, 0.10 µM, and 0.11 µM. The absorbance of the generated ABTS•+ in ultrapure water, underground water, and reservoir water was quite stable within 30 min. Moreover, the proposed Fenton-ABTS method could be used for monitoring the variations of H2O2 concentration during the oxidative decolorization of RhB in alkali-activated H2O2 system.

Salmonella enterica Serovar Typhimurium Interacts with CD209 Receptors To Promote Host Dissemination and Infection.

Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, S Typhimurium was able to bind human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (hCD209a), an HIV receptor that promotes viral dissemination by hijacking antigen-presenting cells (APCs). In this study, we showed that S Typhimurium interacted with CD209s, leading to the invasion of APCs and potentially the dissemination to regional lymph nodes, spleen, and liver in mice. Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection. Thus, we propose that similar to HIV, S Typhimurium may also utilize APCs via interactions with CD209s as a way to disseminate to the lymph nodes, spleen, and liver to initiate host infection.

Spectrophotometric determination of trace hydrogen peroxide via the oxidative coloration of DPD using a Fenton system.

A low-cost and environmentally-friendly spectrophotometric method for hydrogen peroxide (H2O2) determination based on the oxidative coloration reaction of N,N'-diethyl-p-phenylenediamine (DPD) via the Fenton reactions in aqueous water was established. The generated pink radical cation (DPD+) showed maximum absorption at 551 nm. Importantly, under the optimal conditions (pH 3.0, 20 mM DPD, 1.5 mM Fe(II) and reaction time of 45 s), the increase in absorbance at 551 nm for DPD+ generation was linear with respect to the addition of H2O2 (0-12 µM). The sensitivity and the detection limit of the proposed Fenton-DPD method for H2O2 determination at 551 nm were (2.55 ±â€¯0.01) × 104 M-1 cm-1 and 0.05 µM, respectively. The stoichiometric factor for the reaction of H2O2 with DPD was 1:1.18. The absorbance of the generated DPD+ was found to be stable in different types of water within 20 min. Moreover, the proposed Fenton-DPD method could be used for the analysis of the trace H2O2 in rainwater and determine the rate constants that involved H2O2 with high accuracy.

Effective degradation of Orange G and Rhodamine B by alkali-activated hydrogen peroxide: roles of HO2- and O2·.

Advanced oxidation processes offer effective solutions in treating wastewater from various industries. The process of alkali-activated hydrogen peroxide (H2O2) was superior for the treatment of alkaline dye wastewater because no additional reagents were required except H2O2. However, an important and interesting phenomenon had been observed that the primary reactive species were found different for degrading organic pollutants with the process of alkali-activated H2O2. Azo dye of Orange G (OG) and triphenylmethane dye of Rhodamine B (RhB) were chosen as the target organic pollutants. The influences of various parameters on OG and RhB degradation by alkali-activated H2O2 were evaluated. Furthermore, different scavengers, including ascorbic acid, methanol, t-butanol, isopropyl alcohol, furfuryl alcohol, and nitro blue tetrazolium, have been tested to identify the active species involved in dye degradation, and it was found that O2·- was mainly responsible for degrading OG, while HO2- anion was the primary oxidant for degrading RhB.

Murine SIGNR1 (CD209b) Contributes to the Clearance of Uropathogenic Escherichia coli During Urinary Tract Infections.

Uropathogenic Escherichia coli (UPEC), a Gram-negative bacterial pathogen, is a major causative agent of urinary tract infections (UTIs). However, the molecular mechanisms of how UPEC causes infections have not been determined. Recent studies indicated that certain enteric Gram-negative bacteria interact with and hijack innate immune receptors DC-SIGN (CD209a) and SIGNR1 (CD209b), often expressed by antigen-presenting cells (APCs), such as macrophages, leading to dissemination and infection. It was not known whether UPEC could utilize DC-SIGN receptors to promote its infection and dissemination similarly to the enteric pathogens. The results of this study reveal that UPEC interacts with CD209-expressing macrophages and transfectants. This interaction is inhibited by anti-CD209 antibody, indicating that CD209s are receptors for UPEC. Additionally, in contrast to the results of previous studies, mice lacking SIGNR1 are more susceptible to infection of this uropathogen, leading to prolonged bacterial persistence. Overall, the results of our study indicate that the innate immune receptor CD209s participate in the clearance of UPEC during UTIs.

Spectrophotometric determination of trace permanganate in water with N,N-diethyl-p-phenylenediamine (DPD).

A sensitive spectrophotometric method (the N,N-diethyl-p-phenylenediamine (DPD) method) was established for the determination of trace permanganate concentration (0-10 µM) in water. The DPD method was based on the oxidative coloration reaction where permanganate could oxidize DPD to form the red colored DPD radical (DPD•+) with a second-order rate constant of 2.96 × 104 M-1 s-1 at pH 6 (50 mM phosphate buffer). The generated DPD•+ could be quantitatively measured at 551 nm using an UV-Vis spectrophotometer. There was a good linear relationship (R2 = 0.999) between the absorbance of DPD•+ and permanganate concentration. The DPD method was highly sensitive, and the absorbance of generated DPD•+ at 551 nm was as high as 5.70 × 104 cm-1 per M (mol L-1) of permanganate. The reaction of permanganate with DPD in the pH range of 4.0-8.0 had a stoichiometric coefficient of 1:2.71. The residual absorbance of DPD•+ in ultrapure water and natural waters was fairly stable for 30 min. Limits of detection of the proposed DPD method in ultrapure water and natural waters were calculated to be as low as 0.010 µM and 0.017 µM, respectively. Moreover, trace permanganate concentrations of 0.04 and 0.10 µM were found in natural waters and wastewater by the proposed DPD method. Additionally, the DPD method could be applied to measure the second-order rate constants of the reaction of permanganate with phenol at pH 7.0 (28.2 M-1 s-1).

Multi-wavelength spectrophotometric determination of hydrogen peroxide in water with peroxidase-catalyzed oxidation of ABTS.

In this study, a new spectrophotometric method was proposed for the measurement of hydrogen peroxide (H2O2) in aqueous solutions. The method was based on the peroxidase (POD)-catalyzed reaction in which 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was oxidized to form the stable green radical (ABTS+). The generated ABTS+ could be determined spectrophotometrically. The absorbance of the generated ABTS+ at 415 nm, 650 nm, 732 nm and 820 nm were linear with H2O2 concentrations in the range of 0-40 µM. The sensitivities of the proposed ABTS method for H2O2 determination at 415 nm, 650 nm, 732 nm and 820 nm were 6.29 × 104 M-1 cm-1, 2.00 × 104 M-1 cm-1, 2.54 × 104 M-1 cm-1 and 1.89 × 104 M-1 cm-1, respectively. The oxidation of ABTS to generate ABTS+ in POD-catalyzed H2O2 system at pH 6.0 was so fast that the determination time of the ABTS method was as short as 0.5 min. The stoichiometry of the reaction of H2O2 and ABTS in the presence of POD was calculated as nearly 1:2 (1:1.92). The residual absorbance of the generated ABTS+ was also found to be stable within 30 min in natural waters. Low H2O2 concentration in rainwater could be both measured by the ABTS method and the DPD method with high accuracy, however, H2O2 concentration in wastewater contained rhodamine B could only be accurately measured with the ABTS method at 732 nm. Moreover, waste solutions after H2O2 analysis with the proposed ABTS method were non-hazardous towards E. coli.

Opacity proteins of neisseria gonorrhoeae in lipooligosaccharide mutants lost ability to interact with neutrophil-restricted CEACAM3 (CD66d).

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.

Inhibition of melanoma cell proliferation by targeting Wnt/ß-catenin pathway through Sox4 RNA interference.

The effect of siRNA-mediated Sox4 gene silencing on Wnt/ß-catenin signaling pathway of human malignant melanoma cell line A375 was investigated. Two types of dsRNA targeting Sox4 were constructed and transfected into A375 cells, and untreated cells and cells transfected with scramble RNA were used as blank control and negative control respectively. The expression levels of mRNA and protein of Sox4, Wnt3a, ß-catenin and Wnt/ß-catenin signaling target gene Survivin were detected after real-time PCR and Western blot respectively. MTT assay was used to measure cell proliferation after Sox4 knockdown. ß-catenin/TCF transcription reporter assay was used for assessing Wnt/ß-catenin signaling pathway activity. Our results showed that the two types of Sox4 siRNA were transfected into A375 cells successfully. As compared with untreated cells, Sox4 siRNAs had no significant influence on Wnt3a expression, and Sox4 siRNAs led to the decrease of ß-catenin at protein level. Wnt/ß-catenin signaling pathway activity was inhibited significantly. As a target of Wnt/ß-catenin signaling, Survivin was decreased at both mRNA and protein levels, and cell proliferation was attenuated. Our study suggests that Sox4 may play an important role in Wnt/ß-catenin signaling pathway in human malignant melanoma cells by regulating ß-catenin protein level, indicating that Sox4 is involved in the progression of malignant melanoma through Wnt/ß-catenin signaling pathway.

Inhibition of Melanoma Cell Proliferation by Targeting Wnt/β-catenin Pathway through Sox4 RNA Interference / 华中科技大学学报（医学）（英德文版）

The effect of siRNA-mediated Sox4 gene silencing on Wnt/β-catenin signaling pathway of human malignant melanoma cell line A375 was investigated.Two types of dsRNA targeting Sox4 were constructed and transfected into A375 cells,and untreated cells and cells transfected with scramble RNA were used as blank control and negative control respectively.The expression levels of mRNA and protein of Sox4,Wnt3a,β-catenin and Wnt/β-catenin signaling target gene Survivin were detected after real-time PCR and Western blot respectively.MTT assay was used to measure cell proliferation after Sox4 knockdown.β-catenin/TCF transcription reporter assay was used for assessing Wnt/β-catenin signaling pathway activity.Our results showed that the two types of Sox4 siRNA were transfected into A375 cells successfully.As compared with untreated cells,Sox4 siRNAs had no significant influence on Wnt3a expression,and Sox4 siRNAs led to the decrease of β-catenin at protein level.Wnt/β-catenin signaling pathway activity was inhibited significantly.As a target of Wnt/β-catenin signaling,Survivin was decreased at both mRNA and protein levels,and cell proliferation was attenuated.Our study suggests that Sox4 may play an important role in Wnt/β-catenin signaling pathway in human malignant melanoma cells by regulating β-catenin protein level,indicating that Sox4 is involved in the progression of malignant melanoma thromgh Wnt/β-catenin signaling pathway.