Candida causes recurrent vulvovaginal candidiasis by forming morphologically disparate biofilms on the human vaginal epithelium.

Background: Recurrent vulvovaginal candidiasis (RVVC) is a recalcitrant medical condition that affects many women of reproductive age. The importance of biofilm formation by Candida in RVVC has been recently questioned. This study aimed to elucidate the fundamental growth modes of Candida in the vagina of patients with RVVC or sporadic vulvovaginal candidiasis (VVC) and to assess their roles in the persistence of RVVC. Methods: Vaginal tissues were sampled from twelve patients clinically and microbiologically diagnosed as RVVC or VVC at a post-antifungal-treatment and asymptomatic period. High-resolution scanning electron microscopy, fluorescence in situ hybridization in combination with Candida-specific 18S rRNA probes and viable fungal burden were used to qualitatively and quantitatively evaluate Candida growth in the human vagina. The presence of Candida biofilm extracellular polymeric substances was examined using confocal laser scanning microscopy and biopsy sections pre-stained with Concanavalin A. Histopathological analysis was carried out on infected vaginal tissues stained with hematoxylin and eosin. Lastly, the susceptibility of epithelium-associated Candida biofilms to fluconazole at the peak serum concentration was evaluated. Results: Candida species grew on the vaginal epithelium of RVVC patients as morphologically disparate biofilms including monolayers, microcolonies, and macro-colonies, in addition to sporadic adherent cells. Candida biofilm growth on the vaginal epithelium was associated with mild lymphocytic infiltration of the vaginal mucosa. These epithelium-based Candida biofilms presented an important characteristic contributing to the persistence of RVVC that is the high tolerance to fluconazole. Conclusions: In summary, our study provides direct evidence to support the presence of Candida biofilms in RVVC and an important role of biofilm formation in disease persistence.

Antimicrobial Treatment on a Catheter-Related Bloodstream Infection (CRBSI) Case Due to Transition of a Multi-Drug-Resistant Ralstonia mannitolilytica from Commensal to Pathogen during Hospitalization.

Despite its commonly overlooked role as a commensal, Ralstonia mannitolilytica becomes an emerging global opportunistic human pathogen and a causative agent of various infections and diseases. In respiratory illnesses, including cystic fibrosis and chronic obstructive pulmonary disease (COPD), R. mannitolilytica is also identified presumably as colonizer. In this study, one distinctive clone of R. mannitolilytica was firstly identified as colonizer for the first 20 days during hospitalization of a patient. It was then identified as a causative agent for catheter-related bloodstream infection with negative identification after effective treatment, verifying its transition from commensal to pathogen. In conclusion, we provide convincing evidence that during hospitalization of a patient, R. mannitolilytica transitioned from commensal to pathogen in the respiratory tract leading to catheter-related bloodstream infection (CRBSI).

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Antioxidant Profile of 1-Monocaffeoyl Glycerol in Lipophobic/Lipophilic Media.

Oxidative stress has been generally considered as one trigger of organism imbalance, resulting in lipid peroxidation, DNA damage and protein oxidation, which could be relieved by antioxidant supplement or endogenous antioxidant system. In present study, 1-monocaffeoyl glycerol (1-MCG), an amphipathic caffeic acid natural derivative, was enzymatically synthesized by Lipozyme 435, and its antioxidant profile in both lipophilic and lipophobic media was evaluated. The 1-MCG was identified by HPLC-UV, HPLC-ESI-MS, and 1 H/13 C-NMR. Subsequently, antioxidant assays in lipophilic (DPPH assay) and lipophobic (ABTS, ORAC, erythrocyte hemolysis, ROS, MDA, and GPx assays) systems were explored. The better and lasting DPPH· and ABTS+· inhibitions of 1-MCG than caffeic acid (CA) were related to its better solubilities in ethanol/water media and electron transfer ability. ORAC results suggested the radical scavenging activities of 1-MCG (5 to 40 µM) were higher than Trolox. Furthermore, the effectiveness of 1-MCG against AAPH-induced erythrocytes oxidation indicated that 1-MCG can effectively inhibit hemolysis. ESEM was also applied to verify the hemolysis inhibition and morphology preservation abilities of 1-MCG. Besides, results showed 1-MCG was able to prevent ROS from invasion, reduce production of MDA, up-regulated GPx activity, terminate lipid peroxidation, and maintain the integrity of the structure and function of erythrocytes. PRACTICAL APPLICATION: As an amphiphilic caffeic acid derivative, 1-monocaffeoyl glycerol was synthesized, purified, and identified. 1-Monocaffeoyl glycerol could significantly eliminate radicals including DPPH·, ABTS+· , and AAPH in ethanol, water, and PBS system, respectively. 1-Monocaffeoyl glycerol could protect erythrocyte from AAPH induced hemolysis.

Effect of ultrasonic field on the enzyme activities and ion balance of potential pathogen Saccharomyces cerevisiae.

OBJECTIVES: This study aimed at investigating the enzyme activities and ion concentrations in potential pathogen S.cerevisiae upon ultrasonic treatment. METHODS: The activities of ATPase and antioxidase were identified by ATPase, SOD, and CAT assay kits following the instructions. Extracellular Ca2+ and K+ concentrations were determined in an atomic absorption spectrometer with calcium and potassium hollow-cathode lamps as radiation sources. RESULTS: SOD and CAT activities were enhanced by relatively low ultrasonic power at early time points and reduced to lower levels. Total ATPase, Na+/K+-ATPase, and Ca2+/Mg2+-ATPase activities were reduced by ultrasonic field, with higher reducing rate at stronger ultrasonic power and early time points. In addition, ultrasonic field disturbed the Ca2+ and K+ balances in S.cerevisiae cells. CONCLUSIONS: Ultrasonic field resulted in the reduce even the lost of S.cerevisiae cell viability.

Complete Sequence of pCY-CTX, a Plasmid Carrying a Phage-Like Region and an ISEcp1-Mediated Tn2 Element from Enterobacter cloacae.

A plasmid pCY-CTX carrying a phage-like backbone from an extensively drug-resistant Enterobacter cloacae strain Guangzhou-ECL001 (previously known as CY01) was identified in this study. By Illumina MiSeq 2 × 250-bp paired-end sequencing, de novo assembly, and PCR, full sequence of pCY-CTX was obtained. Plasmid pCY-CTX was a circular plasmid with a length of 116,700 bp, harboring 136 putative open reading frames with the average G + C content of 50.8%. The backbone of pCY-CTX showed high identity to previously reported phage-like plasmid pHCM2 and phage SSU5. In addition, pCY-CTX contained a distinctive ISEcp1-mediated Tn2 region with two resistance genes blaTEM-1 and blaCTX-M-3. Transposition unit "ISEcp1- blaCTX-M-3- orf477" was inserted into the Tn2 structure, dividing Tn2 into two parts. This represents the first identification of a plasmid carrying a phage-like backbone and a distinctive ISEcp1-mediated Tn2 region within blaTEM-1 and blaCTX-M-3 in clinical E. cloacae. The finding of phage-like regions located in plasmids provides a new perspective in gene transfer associated with antimicrobial resistance.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Analysis on pathogenic and virulent characteristics of the Cronobacter sakazakii strain BAA-894 by whole genome sequencing and its demonstration in basic biology science.

Cronobacter sakazakii is an opportunistic pathogen responsible for necrotizing enterocolitis, meningitis and septicaemia especially to infant and neonate, with high lethality ranging in 40%-80%. This strain is able to survive in infant milk formula and possesses capability of pathogenicity and virulence, biofilm formation, and high resistance to elevated osmotic, low pH, heat, oxidation, and desiccasion. This study is aims to investigate the molecular characteristics of Cronobacter sakazakii BAA 894, including mechanisms of its invasion and adherence, biofilm formation, unusual resistance to environmental stress employing whole genome sequencing and comparative genomics. Results in this study suggest that numerous genes and pathways, such as LysM, Cyx system, luxS, vancomycin resistance pathway, insulin resistance pathway, and sod encoding superoxide dismutase for the survival of C. sakazakii in macrophages, contribute to pathogenicity and resistance to stressful environment of C. sakazakii BAA 894.

Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates.

Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs) in foods. Peptide-enriched drinks (PEDs) are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr). In this study we determined free-form pyrraline (Free-Pyr) and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH), soy protein hydrolysate (SPH) and collagen protein hydrolysate (CPH). A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE). The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD) of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent) of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent) of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

Formation of Peptide Bound Pyrraline in the Maillard Model Systems with Different Lys-Containing Dipeptides and Tripeptides.

Peptide-bound advanced glycation end-products (peptide-bound AGEs) can be formed when peptides are heated with reducing saccharides. Pyrraline is the one of most commonly studied AGEs in foods, but the relative importance of the precursor peptide structure is uncertain. In the present study, model systems were prepared by heating peptides with glucose from 60 °C to 220 °C for up to 65 min, and the amounts of peptide-bound pyrraline formed were monitored to evaluate the effect of the neighboring amino acids on the peptide-bound pyrraline formation. The physico-chemical properties were introduced to explore the quantitative structure-reactivity relationships between physicochemical properties and peptide bound formation. 3-DG content in dipeptide-glucose model system was higher than that in the corresponding tripeptide-glucose model systems. Dipeptides produced higher amounts of peptide-bound pyrraline than the corresponding tripeptides. The peptide-bound pyrraline and 3-DG production were influenced by the physico-chemical properties of the side chain of amino acids adjacent to Lys in the following order: Lys-Leu/glucose > Lys-Ile/glucose > Lys-Val/ glucose > Lys-Thr/glucose > Lys-Ser/glucose > Lys-Ala/ glucose > Lys-Gly/glucose; Lys-Leu-Gly/glucose > Lys-Ile-Gly/glucose > Lys-Val-Gly/glucose > Lys-Thr-Gly/glucose > Lys-Ser-Gly/glucose > Lys-Ala-Gly/glucose > Lys-Gly-Gly/glucose. For the side chain of amino acids adjacent to Lys in dipeptides, residue volume, polarizability, molecular volume and localized electrical effect were positively related to the yield of peptide bound pyrraline, while hydrophobicity and pKb were negatively related to the yield of peptide bound pyrraline. In terms of side chain of amino acid adjacent to Lys in tripeptides, a similar result was observed, except hydrophobicity was positively related to the yield of peptide bound pyrraline.

In vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural.

5-HMF is widely presented in foods and produced through the degradation of hexoses and Maillard reaction during heat treatment of foods containing reducing sugars and amino acids in an acid environment. However, controversial conclusions on the biological effects of 5-HMF have been drawn in previous studies. Therefore, the main aim of this study was to investigate the antioxidant and antiproliferative activities of 5-HMF. The 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) assay, the 1,1-diphenyl-2-picryhydrazyl (DPPH) assay, and the hemolysis assay induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were performed to evaluate the antioxidant capacity of 5-HMF. The results showed that 5-HMF exhibited novel antioxidant activity by scavenging the ABTS and DPPH free radicals and inhibited the AAPH-induced hemolysis in a dose-dependent manner. In the hemolysis assay, the reduction of ROS and MDA contents and the increase in enzyme activities of SOD, CAT, and GPx were found in erythrocytes pretreated with 5-HMF, which demonstrated that 5-HMF could prevent the peroxidation from the source to protect the erythrocytes. The morphological changes of erythrocytes was also verified by observation using atomic force microscopy. The inhibitory effect of 5-HMF on human cancer cell proliferation was investigated by MTT assay, flow cytometric analysis, and the TUNEL and DAPI costaining assay. The results showed that 5-HMF displayed higher antiproliferative activity on human melanoma A375 cells than other cell lines. Further investigation on the action mechanisms revealed that 5-HMF could induce A375 cell apoptosis and G0/G1 cell cycle arrest. The A375 cell apoptosis that 5-HMF induced was characterized by a TUNEL and DAPI costaining assay. These findings suggest that 5-HMF could be developed as a novel natural antioxidant with potential applications in cancer chemoprevention.

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil.

The effectiveness of ultrasonic-assisted extraction (UAE) of pomegranate seed oil (PSO) was evaluated using a variety of solvents. Petroleum ether was the most effective for oil extraction, followed by n-hexane, ethyl acetate, diethyl ether, acetone, and isopropanol. Several variables, such as ultrasonic power, extraction temperature, extraction time, and the ratio of solvent volume and seed weight (S/S ratio) were studied for optimization using response surface methodology (RSM). The highest oil yield, 25.11% (w/w), was obtained using petroleum ether under optimal conditions for ultrasonic power, extraction temperature, extraction time, and S/S ratio at 140 W, 40 °C, 36 min, and 10 ml/g, respectively. The PSO yield extracted by UAE was significantly higher than by using Soxhlet extraction (SE; 20.50%) and supercriti cal fluid extraction (SFE; 15.72%). The fatty acid compositions were significantly different among the PSO extracted by Soxhlet extraction, SFE, and UAE, with punicic acid (>65%) being the most dominant using UAE.

Overexpression of Pim-1 in bladder cancer.

BACKGROUND: Pim-1 is a serine-threonine kinase which promotes early transformation, cell proliferation and cell survival during tumorigenesis. Several studies have demonstrated that Pim-1 kinase play a role in different cancer types, however, the function of Pim-1 in bladder cancer is poorly understood. METHODS: Expression and localization of Pim-1 in human normal and malignant bladder specimens were examined by Immunohistochemistry and Pim-1 staining score was compared with several clinicopathologic parameters. To further demonstrate the biological function of Pim-1 in bladder cancer, its expression was validated in five bladder cancer cell lines by western blot and immunohistochemistry analyses. Subsequent knockdown of Pim-1 was achieved by lentivirus encoding small interfering RNA, and the effect of Pim-1 on bladder cell survival and drug sensitivity were further assessed by colony formation and cell proliferation assays. RESULTS: When compared with normal epithelium, Pim-1 was overexpressed in bladder cancer epithelium, and the expression level was higher in invasive bladder cancer than Non-invasive bladder cancer specimens. Pim-1 was also detected in all the bladder cancer cell lines examined in our study. Moreover, the knockdown of Pim-1 significantly inhibited bladder cancer cell growth and also sensitized cells to chemotherapeutic drugs in vitro. CONCLUSIONS: Our results in this study suggest that Pim-1 may play a role in bladder cancer initiation and progression. Since Pim-1 is also involved in bladder cancer cell survival and drug resistance, Pim-1 is a potential candidate for targeted therapy in bladder cancer.

Frequency upconversion of multiple RF signals using optical carrier suppression for radio over fiber downlinks.

We propose and analyze a technique of an optical carrier transmitting two RF signals using optical carrier suppression. A single optical Mach-Zehnder modulator is used for both optical carrier suppression and signal modulation, and optical carrier suppression modulation is also used for frequency conversion of RF signals. This work shows that in contrary to the case of an optical carrier transmitting a single RF signal with optical carrier suppression where stronger optical carrier suppression improves the upconverted RF signal, weaker optical carrier suppression is preferred for an optical carrier transmitting two RF signals due to nonlinear distortion because the nonlinear distortion is reduced by using weaker optical carrier suppression. We find that the usable range of optical carrier suppression ratio is from 10 to 18 dB for RF signal upconverted to 20 GHz and beyond, and the best optical carrier suppression ratio is around 10 dB. We verify the concept and analysis with experiment. In experiment, we used two RFs at 6 and 18 GHz transmitting two 750 Mb/s signals. The experiment for the first time demonstrated that an optical carrier can transmit two RF signals using optical carrier suppression and showed that upconverted RF signals are degraded by nonlinear distortion, particularly for upconverted RF signal at 12 GHz, i.e. the RF signal at the lower frequency.