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FIG. 4.

Subtractive microarray approach to assay autoaggregation. As described in the text, the effect of autoaggregation was determined using two separate conditions: “SDM +/− saliva” and “SDM + saliva +/− lysine.” To determine whether a gene is either affected by aggregation or differences in medium composition, the responses from both conditions were compared. Only the gene responses that were similar between the two microarray experiments were considered to be due to autoaggregation, whereas gene responses that were specific to either of the two microarray datasets were attributed to factors unrelated to autoaggregation. Arrows underneath each microarray condition point to the total number of affected genes from each experiment. A comparison of the two datasets identified the shared gene responses, which were attributed to aggregation. The total number of genes within each geneset is listed.

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FIG. 5.

Phase-contrast images of cells used for microarray. Phase-contrast images of the same cultures used for RNA extraction and microarray were taken just prior to RNA extraction. The total magnification is ×1,000. The medium compositions are as follows: SDM (A), SDM plus 25% saliva (B), and SDM plus 25% saliva plus 50 mM lysine (C).

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Selected genes from the microarray data set

The overall response to aggregation is summarized in Table 3 . When grouping genes by basic functional categories, the greatest number of genes affected by cell-cell contact involves transport and/or substrate binding. Of these, a large portion comprises putative permeases of a variety of nutrients, as well as putative phosphoenolpyruvate-dependent sugar phosphotransferase system components, which presumably bind certain sugars. Similarly, numerous putative amino acid and peptide transporters were induced as well. In addition to genes involved in nutrient acquisition, three separate putative Na + /H + antiporters were induced (FN0650, FN1422, and FN2077), as well as an operon predicted to encode an efflux pump used to confer resistance to multiple antimicrobials (FN0515 and FN0516). Given the large number of transporters affected, it is not surprising that many genes involved in energy and/or intermediate metabolism were affected as well. From these genes, a large portion (at least 10) can be attributed to the metabolism of ethanolamine. The genes required for this ability are all clustered and arranged into two to three large operons of “ eut” genes (FN0078 to FN0090).

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Summary of affected genes

Real-time RT-PCR confirmation of microarray results. Based upon the microarray data, it appeared that aggregation had a similar effect upon gene expression when measured under both assay conditions (+/− saliva and saliva +/− lysine). To further confirm these data, we analyzed the RNA used for the microarray studies via real-time RT-PCR. We tested a representative subset of the total microarray data set based upon chromosome location, putative gene function, and the magnitude of the gene expression change. As shown in Fig. Statement Clutch Abstract on silk by VIDA VIDA Cheap Original ElBVgtkKa
, we found that most of the genes were indeed affected quite similarly between the +/− saliva and saliva +/− lysine experiments. The exception was FN1900, which was one of only four genes found by microarray to have lower expression upon aggregation. From the real-time RT-PCR analysis, it appeared that this gene is lower expressed only in the saliva +/− lysine condition, which suggests that this effect is probably unrelated to aggregation. However, it should be noted that in the microarray data set, the decreased expression of FN1900 was much more apparent in the saliva +/− lysine samples (>4-fold) compared to the +/− saliva samples (<2-fold) (Table 2 ). Therefore, the trend in FN1900 gene expression was still consistent between the microarray and real-time PCR analyses. Overall, we found that the degree of differential gene expression in the real-time PCR analysis corresponded reasonably closely to the microarray values with the main exceptions being FN1988 and FN1989, which seemed to have been underestimated by microarray analysis. However, the trends of expression were again consistent, since these genes had the greatest increases in both the microarray data set and the real-time PCR experiments. In addition, there seemed to be little evidence that the microarray had overestimated gene expression changes. Thus, it appears that the microarray data are likely to be an accurate representation of the overall trends in gene expression and in many cases are also likely to be a close reflection of the gene expression changes as measured by real-time RT-PCR.

As both species possess individual genomes and utilize unique metabolic pathways, it is likely that as well as the metabolic production of H 2 O 2 by S. pneumoniae , there exists a myriad of biological factors that may impact the growth of one of the species. These factors include metabolic by-products produced by one of the species, which can be beneficial or detrimental to the other species. The metabolic by-products generated are in turn dependent on the environmental conditions, nutrient availability and physical conditions of the environment.

Recently, it has been observed that in a batch coculture model of S. pneumoniae and H. influenzae growth, S. pneumoniae was able to outcompete H. influenzae (A. Tikhomirova S.P. Kidd, unpublished observation, 2012). This effect was not dependent on the strains used and was not a result of H 2 O 2 -mediated competition as an spxB isogenic mutant of S. pneumoniae also inhibited H. influenzae planktonic and biofilm growth. Importantly, this growth inhibition effect was time dependent, where H. influenzae cell death occurred only after the entering of both species into stationary phase. This growth inhibition effect appeared to be a result of a pH change occurring in the S. pneumoniae growth after reaching stationary phase (A. Tikhomirova S.P. Kidd, unpublished data, 2013). Hence, it appears that by modifying the pH of the immediate environment during certain growth stages, S. pneumoniae can diminish the viability of H. influenzae . Hence, H. influenzae may be more adapted for survival and potentially biofilm formation in an environment where pH is elevated. This is supported by the finding that the type IV pili important for H. influenzae biofilm formation were expressed in a chemically defined medium at pH 8.5–9.0, but not 7.2 (Bakaletz et al. , 2005 ). The pH of the middle ear environment has not been extensively studied; however, it has been shown that the pH of middle ear effusions of OM patients with chronic secretory forms of OM extends to a basic pH that ranges from pH 7–9 (Nuutinen et al. , 1993 ; Wezyk Makowski, 2000 ). If S. pneumoniae lowers the pH during a multispecies infection, H. influenzae must have mechanisms to regulate the pH to maintain a neutral or basic pH of the environment. One pH regulatory mechanism in H. influenzae is urease. The H. influenzae urease is a nickel-dependent enzyme, which is involved in catalysing the hydrolysis of urea to ammonia and carbon dioxide (Mobley et al. , 1995 ). In other bacteria, the urease functions to raise the pH of the environment, allowing bacterial survival at low pH environments (Ferrero Lee, 1991 ; Stingl et al. , 2002 ). In a chinchilla model of OM, it has been demonstrated that the ureH , a homologue of a gene requires for expression of an active urease in Helicobacter pylori , was upregulated in the chinchilla middle ear (Mason et al. , 2003 ). Additionally, in human chronic obstructive pulmonary disease, antibodies against H. influenzae urease increased with exacerbations of the disease, indicating urease expression in chronic diseases of the respiratory tract (Murphy Brauer, 2011 ). The ure operon was also found to be more prevalent in H. influenzae isolates from OM or COPD than from isolates obtained from throats of healthy individuals (Zhang et al. , 2013 ). It would be likely that if S. pneumoniae growth dramatically reduces the pH of the environment (A. Tikhomirova S.P. Kidd, unpublished data), then for a coculture growth within the middle ear, acidification of the environment would be prevented or shifted to a more alkalizing state that would facilitate survival, type IV pili expression and a multispecies biofilm formation. Hence, in chronic forms of OM that involve multispecies infections, H. influenzae may require urease or other systems to maintain the pH at a neutral or slightly basic state, which is consistent with the clinical observations for the presence of urease.

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La nueva Volkswagen Teramont buscará ser protagonista en su campo de batalla con atributos de espacio interior, seguridad, diseño y atractivos planes de financiamiento

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ENSENADA, BC.- Volkswagen acudió al viejo refrán que dice “Más vale tarde que nunca”, para presentar en el mercado mexicano la nueva Teramont, el modelo con el que la firma alemana por fin entra a competir en el segmento de las camionetas medianas de tres filas de asientos contra productos como Honda Pilot, Toyota Highlander, Mazda CX-9, y Ford Explorer entre otras.

Este modelo estará a la venta en las agencias de la marca a principios de agosto y llegará desde Chattanooga, Tennessee, donde es construida sobre la plataforma MQB, la cual recibe carrocerías más pequeñas como la del Golf, Jetta o Tiguan, pero con una distancia entre ejes más larga (2.97 metros) que le permite ser el vehículo con mayor espacio en la tercera fila del segmento, según la compañía Mide 5.03 metros de largo, lo que le da alrededor de seis centímetros más de espacioque la mayoría de sus competidores.

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“Teramont es muy importante para nosotros porque se convierte en nuestro representante más grande, es una camioneta con capacidades importantes en almacenamiento, tren motriz, tecnologíay de seguridad.

“Fue presentada hace dos años para el mundo y ha tenido mucho éxito en países como Estados Unidos, Canadá, Rusia y en países árabes. Ahora llega a México donde también esperamos cosas importantes”, comentó Edgar Estrada, director general de Volkswagen de México.

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“Uno de los puntos relevantes en los que trabajamos fuerte y de la mano de nuestra financiera de marca, es en los planes de financiamiento. Queremos tener esquemas muy atractivos” señaló.

De inicio la marca anunció que ofrecerá esta camioneta con un plan de financiamiento a 36 meses y con mensualidades desde 9,999 pesos.

Teramont estará disponible en cuatro versiones: Trendline ($669,990), Comfortline ($724,990), Comfortline Plus ($779,990) y Highline ($859,990).

Figure 1. Abundance of in rectal mucosal biopsies from adenoma cases and non-adenoma controls.

qPCR results show that Fusobacterium is more abundant in cases than controls.


Table 1. Characteristics of Study Participants.

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Table 2. Association between abundance and colorectal adenomas.

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We Observed that Fusobacterium was over-represented in adenoma cases compared to non-adenoma controls, therefore, we performed histological evaluation by Fluorescence in situ Hybridization (FISH) using a Fusobacterium -specific probe to localize Fusobacterium in colonic mucosal tissue sections ( Fig. 2a and 2b ). The results show that Fusobacterium was localized in the mucus layer above the epithelium as well as within the colonic crypts. A general bacterial probe was also used as a positive control ( Orange 100% Original Leopard Bikini Top Leopard Pukas Surf Best Seller For Sale Cheap Sale For Cheap Free Shipping Sneakernews Sale Shop For qxNGCQ
). Results confirm the presence of bacteria in the mucus layer.

Figure 2. Representative fluorescence hybridization targeting in colorectal mucosal biopsy sections using bacterial 16S rRNA probes.

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are composite images of Cy3 and DAPI views of sections hybridized with a Fusobacterium -specific probe. Fusobacterium species is localized within the mucus layer of colorectal sections (A) 20X and 40X. Fusobacterium species is localized within the crypts of colorectal section (B) 20X and 40X. Fig. 2C (20X and 40X) is a positive control and shows sections stained with general bacteria probe (Eub 388). General bacteria, including most Eubacteria species, are localized to the mucus layer above the epithelium. White arrows point to bacteria either in mucus layer above the colonic epithelium or within the crypt.


Correlation of local inflammatory cytokine gene expression and Fusobacterium species abundance was analyzed separately for adenoma cases and non-adenoma controls. Analysis of cytokines IL-6, IL-10, IL-12, IL-17 and TNF-α and Fusobacterium was observed to have a significant positive correlation with local inflammation in cases, but not controls ( fur collar scarf Blue Cara Mila Outlet Sale Online Sale Geniue Stockist Best Cheap Price For Nice For Sale Hyper Online uUR7J2
). A significant positive correlation was found between abundance of Fusobacterium species and IL-10 (r  = 0.443 p  = 0.01). The correlation for TNF-α (r = 0.335 p = 0.06) was borderline significant. Although the correlations for IL-6 and IL-17 were positive, they did not reach statistical significance.

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