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1Department of Anatomy and Cell Biology, Hanyang University, College of Medicine, Seoul 133-791, Korea. 2Department of Environmental Biology and Medical Parasitology, Hanyang University, College of Medicine, Seoul 133-791, Korea. Lactobacillus species in the female genital tract are thought to act as a barrier to infection. The vaginal mucosa is populated with microflora, with lactobacilli being the dominant microbes. We examined interactions between Lactobacillus acidophilus and in vitro cultured VECs, because this bacterial strain is frequently recovered from the vagina [7]. An acidic vaginal pH and the presence of lactobacilli are clearly components of the multifaceted antimicrobial defense system that operates in the vaginal fluid, because lactobacilli produce organic acids, including lactic acid [10]. Vaginal floras are dynamic, and the level of lactobacilli used in the present study may not reflect the level of lactobacilli present in the vagina of women. Analysis of pH, optical density (OD600 nm), and number of colony forming units (CFUs) from cultures of L. Hardware GPS-Trace Orange supports 600+ GPS device types You may use your smartphone or any personal GPS-tracker and vehicle controller.
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Giulia Tabanelli1, Pamela Vernocchi1,2, Francesca Patrignani3, Federica Del Chierico2, Lorenza Putignani2,4, Gabriel Vinderola5, Jorge A. Applying sub-lethal levels of high-pressure homogenization (HPH) to lactic acid bacteria has been proposed as a method of enhancing some of their functional properties. Recently, there has been increasing interest in the potential of applying techniques such as pulsed electric field (PEF), high-hydrostatic pressure (HHP), or high-pressure homogenization (HPH) to enhance the survival rate of probiotic strains or to modify their overall functionality in a positive manner. Considering that some probiotic properties are associated with the bacterial cell wall, which is also the principal target of HPH, the aim of this study was to evaluate the effect of sub-lethal HPH treatment on the outermost structures of two strains endowed with probiotic features (Lactobacillus acidophilus DRU and Lactobacillus paracasei A13) using transmission electron microscopy (TEM). The cells in overnight cultures were harvested by centrifugation (8000 g, 10 min, 4°C).
Transmission electron microscopy (TEM) was used to investigate the morphological changes caused by the HPH treatment. The strain viability rate and the aroma profiles of the LAB-containing buttermilk were determined immediately after inoculation and at 15 and 30 days of refrigerated storage. The results of volatile profile analysis of each sample were expressed as the mean values of six independent replicate analyses (conducted on different days) and the data were analyzed using Principal Component Analysis (PCA) using Statistica 6.1 software (StatSoft Italy srl, Vigonza, Italy).
A proteomic approach using MALDI-TOF MS-based protocols was used to investigate the effect of HPH treatment and the subsequent incubation on the peptide profiles of L.
Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. The dominance of lactobacilli over pathogenic anaerobes is positively associated with vaginal health [1], as evidenced by the strong association between reduced numbers of vaginal lactobacilli and common vaginal disorders, including bacterial vaginosis [1] and trichomoniasis [2,3]. An alteration in vaginal pH to less acidic values is strongly associated with vaginal flora changes. Further clarification of this relationship could improve our understanding of Trichomonas and may have implications for the treatment of trichomoniasis. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF 531-2008-1-E00043 and KRF 2009-0074679).
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I can't think of all the things, besidestheshoegaze influence thattheEdge brought opt opt in in here, that can justifytheclaim that U2 made the huge comeback opt opt in quality with this. This means that you will not need to remember your user name and password in the future and you will be able to login with the account you choose to sync, with the click of a button. This page doesn't support Internet Explorer 6, 7 and 8.Please upgrade your browser or activate Google Chrome Frame to improve your experience. Because the principal targets of HPH are the cell-surface structures, the aim of this study was to examine the effect of sub-lethal HPH treatment on the outermost cellular structures and the proteomic profiles of two known probiotic bacterial strains. These strains were chosen based on the results of previous studies that demonstrated their ability to increase certain functional properties in response to HPH treatment (Tabanelli et al., 2012, 2013).
Additionally, the volatile profiles of buttermilk inoculated with treated or control cells during 30 days of refrigerated storage were investigated to evaluate the impact of the sub-lethal HPH treatment on the accumulation of molecules that can impart aromas to the product. Cell counts were obtained by plating the cultures on MRS agar (37°C, 48 h, under aerobic conditions). Solid-phase microextraction and gas-chromatography-mass spectrometry (SPME-GC-MS) were used to detect the aroma compounds as reported by Patrignani et al. Eight replicate MALDI TOF MS Biotyper analyses of each sample were conducted and each replica was considered independently.
However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Trichomoniasis is the most common nonviral sexually-transmitted infection worldwide and an apparent contributor to HIV acquisition [1,2].
For example, Trichomonas, Chlamydia, and Neisseria infections are associated with increases in the vaginal pH [11]. The OD600 nm of cultured media collected from culture flasks was measured using a Beckman spectrophotometer (Beckman, Fullerton, California, USA). MS74 cells were cultured in T75 cell culture flasks (SPL lifesciences, Pocheon, Korea) in the presence or absence of L. Adhesion of lactobacilli results in the formation of a bacterial film on the vaginal epithelium and may contribute to the exclusion of pathogens from the vaginal mucosa. The co-culture system described in this study could therefore be a valuable tool for investigating pathogenesis or for finding compounds that can protect against common vaginal disorders, including bacterial vaginosis and trichomoniasis. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. The association between Trichomonas vaginalis infection and level of vaginal lactobacilli, in nonpregnant women. Competitive exclusion of uropathogens from human uroepithelial cells by Lactobacillus whole cells and cell wall fragments.
Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates. Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora.
Estimation of vaginal probiotic lactobacilli growth parameters with the application of the Gompertz model. Epithelial differentiation promotes the adherence of type 1-piliated Escherichia coli to human vaginal cells. Trichomonas vaginalis: in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes. Mouse intravaginal infection with Trichomonas vaginalis and role of Lactobacillus acidophilus in sustaining infection.
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Moreover, the effect of HPH treatment on the metabolism of probiotic cells within a dairy product during its refrigerated storage was investigated using SPME-GC-MS. Moreover, although the responses varied according to the characteristics of individual strains of lactic acid bacteria (LAB), HPH increased the activity of extracellular or cell wall-associated proteolytic enzymes without having detrimental effects on their viability, confirming their tolerance of moderate pressures.
In particular, the latter authors demonstrated that an HPH treatment applied at 50 MPa modulated the hydrophobicity and auto-aggregation of the treated strains in vitro and modified their interaction with the small-intestinal structures of BALB mice.
Moreover, bacterial profiling analyses of treated and untreated strains were performed using MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) to evaluate the effect of HPH on the cellular peptide profiles.
The cells were subjected to high-pressure homogenization (HPH) at 50 MPa using a PANDA high-pressure homogenizer (Niro Soavi, Parma, Italy).
Prior to performing principal component analysis (PCA)-based hierarchical clustering, each spectrum was subjected to mass adjustment, smoothing, baseline subtraction, Normalization and peak picking.
Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. Trichomonas vaginalis has been associated with multiple morbidities, including pelvic inflammatory diseases, preterm birth, cervical cancer, and infertility as well [1,2]. To determine the number of viable microorganisms, culture suspensions were diluted 104- and 106-fold in sterile distilled water, and then 100 Aµl of these diluted culture suspensions were spread onto MRS agar plates.
Previous studies have shown that the numbers of vaginal lactobacilli are reduced in the presence of a Trichomonas infection, and vice versa [13,14]. These results suggest that lactobacilli contribute to a healthy environment for VECs, thereby facilitating resistance to pathogens, although the exact mechanisms still need to be clarified. Transmission electron microscopy was used to examine the microstructural changes in the outermost cellular structures due to HPH treatment. The HPH-treated cells showed a different behavior in the mouse gut and induced a stronger IgA response compared to those of untreated cells, in strain- and feeding-period-dependent manners. MALDI-TOF MS has been applied to microbial detection because it generates characteristic mass spectra that are unique for each species, permitting identification at the genus and species levels, and potentially, at the strain level (Croxatto et al., 2012).
The dendrogram for a single organism was created using with distance measurements (correlation), linkage (average) and a 300-score threshold values using MALDI Biotyper 3.1 software (Bruker Daltonics GmbH). As shown in Figures 1A,B, cell-wall and inner-membrane structures were clearly visible in the control (0.1 MPa) cells of L. Lactobacilli are believed to protect their hosts from urogenital tract infections via various mechanisms.
After 24 hr culture, the culture flask was examined under a microscope (A—400) (TS100, Nikon, Tokyo, Japan).

In other words, VECs may be protected by the adherence of lactobacilli, and the vaginal ecosystem may be more resistant to pathogens because of the presence of lactobacilli.
Our model co-culture system can potentially be used to identify the factors that maintain a healthy vaginal micro-biota.
These alterations may be involved in the changes in some of the technological and functional properties of the strains that were observed after pressure treatment.
The peptide mixtures derived from acidic hydrolysis were decanted rather than being separated from the insoluble material by centrifugation to avoid possible peptide co-precipitation.
The Pearson's correlation coefficients were calculated using spectral row data using R-Bioconductor to establish the reproducibility of the intra- (replicates) and inter-strain conditional data. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state.
For example, lactobacilli have higher affinity for human uroepithelial cells in vitro and inhibit other pathogenic bacteria from adhering to those cells based on competitive exclusion [4,5]. Consistent with the previous report [12], these pH changes corresponded to the maximum growth rate of the bacterium, as shown in Fig. Which often is sort of still , not quite behind you see, the scenes of you see, the ZooTV tour.
Moreover, the proteomic profiles of the probiotic strains treated with HPH and incubated at 37°C for various periods showed different peptide patterns compared with those of the untreated cells. It is known that when pressure is applied at a sub-lethal level, various cellular responses occur and that the composition of the cellular membrane can change to withstand the exposure to a sub-lethal stress (Russell et al., 1995). The high level of versatility and the speed and accuracy of this methodology played key roles in its adoptation in many fields, including clinical diagnostics, environmental monitoring, and food-quality control.
Immediately after the treatment, the samples were rapidly cooled to 10°C in a water bath. The post-fixed cells were washed using the same buffer and then they were dehydrated for 15 min using the following series of ethanol solutions: 50, 75, 90, and 100%.
Buttermilk was chosen as the dairy medium for this study because previous studies had shown that it supported the growth and the survival of adequate numbers of LAB cells during storage (Burns et al., 2008b).
TEM images of cells of the same strain after HPH treatment at 50 MPa (Figures 1C,D) showed changes in the structures of 70–80% of the cells. They are also capable of co-aggregating with pathogenic bacteria, which may result in the elimination of pathogens [6].
In addition, there were differences in the peaks that were observed in the low-mass spectral region (2000–3000 Da) of the spectral profiles of the control and treated samples.
Although MALDI-TOF MS analysis is an interesting approach to microbial characterization, it has rarely been applied to food-related microorganisms. The dehydrated cells were infiltrated with increasing concentrations of Spurr resin (Agar Scientific, Stansted, Essex, United Kingdom) over 24 h. Peptide mass spectra were acquired using a Microflex MALDI-TOF-MS (Bruker Daltonik GmbH) mass spectrometer that was operated in the linear positive mode at the maximum frequency (20 Hz).
Several studies have demonstrated that lactobacilli adhere to vaginal epithelial cells (VECs). Because high numbers of lactobacilli and a low pH are characteristic of a healthy human vagina only, but not in animals [16], our in vitro model could be used to study the possible roles of Trichomonas virulence factors in reducing the number of lactobacilli as well as any direct harmful effects of the virulence factors on the VECs. Due to pressure treatment, the volatile-molecule profiles of buttermilk inoculated with treated or control cells and stored at 4°C for 30 days exhibited overall changes in the aroma profile and in the production of molecules that improved its sensory profile, although the two different species imparted specific fingerprints to the product. Polymerization of the resin was achieved by heating the samples in an oven at 65°C for 18 h. However, little is known about the effects of lactobacilli adherence to VECs on either the bacteria themselves or VECs.
The results of this study will contribute to understanding the changes that occur in the outermost cellular structures and the metabolism of LAB in response to HPH treatment.
Thin sections (approximately 90 nm thick) were placed on carbon-coated Formvar-covered 300-mesh copper grids for approximately 15 min, rinsed using 20 drops of distilled water, negatively stained using 6–7 drops of 2% aqueous uranyl acetate and then examined using a Philips CM 10 transmission electron microscope. Volatile-peak identification was conducted via computerized matching of the mass spectral data with those for the compounds contained in the Agilent Hewlett-Packard NIST 98 and Wiley vers.
Moreover, the cytoplasm appeared to be compressed and it was detached from the outermost cellular structures (indicated using an arrow in Figure 1D). We therefore investigated the effects of adherence of lactobacilli to in vitro cultured VECs by analyzing the acidity of the culture medium, cell viability, and proliferation in the presence or absence of lactobacilli. The findings of this investigation may contribute to elucidating the relationships between these changes and the alterations of the technological and functional properties of LAB induced by pressure treatment.
The spectral profiles were visualized using FlexAnalysis 3.0 software (Bruker Daltonics GmbH). The effects of pressure treatment on the outermost cellular structures were also visible in the TEM images of L.
This pH value measured in the media from co-cultures is similar to the value measured in healthy vaginas.
The cells were harvested by incubation with 10% trypsin-EDTA solution (GenDEPOT), and then washed twice and resuspended with PBS via centrifugation at 2,000 g at 4a„? for 30 min. This in vitro culture system could contribute to investigation of alternative curative therapies in a number of refractory cases. In particular, these authors reported that these treatments reduced the level of cyclic fatty acids and increased the unsaturation level, leading to modification of the membrane fatty-acid profile in a strain-dependent manner.
The SPME-GC-MS results for each sample at each time point (un-inoculated and untreated buttermilk at 0, 15, and 30 days of storage) were expressed as the mean values of six independent analyses. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. In contrast, there was no change in the pH of the media from MS74 cells cultured alone, as expected (Fig.
Viable cells were counted by staining the cells with trypan blue followed by visualization under a microscope using a hemocytometer (Superior MarienFeld, Bad Mergentheim, Germany). As shown in Figures 2A,B, a continuous, thin, electron-dense layer was visible at the outer edge of the walls of the L. Each value represents the meanA±SD from 3 independent experiments, with each condition tested in triplicate.
The HPH-treatment induced morphological changes observed in the TEM images did not significantly impaired cell viability, as demonstrated by cell counts obtained after HPH treatment. However, these morphological changes could be related to changes in some of the technological and functional properties of the strains observed after pressure treatment that were reported by Tabanelli et al. This co-culture model could therefore potentially be used to mimic vaginal conditions in future in vitro studies.
The discrepancy in OD600 nm values may partly be explained by metabolic wastes produced by the co-cultured MS74 cells.

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