Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli -induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl-L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli -caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5-Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli -induced pathogenicities in a manner dependent on MUC2.
E. coli is the most common gram-negative bacterium that causes neonatal sepsis and meningitis (NSM) [27 , 28 , 47] . Most E. coli -induced neonatal bacterial meningitis cases occur via hematogenous spread  . It is accompanied by the three hallmark features of bacterial meningitis: NF-κB activation, meningitic E. coli invasion, and polymorphonuclear neutrophil migration through the blood-brain barrier (BBB) [10 , 11 , 13 , 51 , 56] . The E. coli -induced NSM generally occurs when bacterial pathogens adhere to intestinal epithelial cells and then cross the intestinal wall into the bloodstream [31 , 32] . Previous studies of humans and animals indicate that the development of NSM is associated with the magnitude of bacteremia, which is essential for pathogen crossing the BBB [31 , 32] .
In recent years, there has been a raising incidence of early onset E. coli infections in very low birth weight neonates. Widespread use of antibiotic may result in an increasing incidence of neonatal infections caused by antibiotic resistance, which has become an evolutionary and ecological problem originating from the response of bacteria to antibiotics  . The ongoing antimicrobial resistance crisis will be certainly enhanced by widespread antibiotic use, leading to the increasing global incidence of infectious diseases that are untreatable with any known antimicrobial agent  . Recently, from clinical observations, probiotic microorganisms have been shown to be useful in preventing or treating certain infectious diseases as well as allergic disorders, including vaginitis, inflammatory bowel disease, and atopic dermatitis. Both prophylactic and therapeutic effects have been observed in children and adults [1 , 3 , 15 , 29 , 46] . As probiotics contribute to the maintenance of ecological balance, the use of probiotics for the prophylaxis of early onset neonatal sepsis or meningitis may overcome the major adverse consequence of widespread antibiotic use, which disturbs the normal microbiota [17 , 20] .
The introduction of probiotic microorganisms ( e.g. , probiotic agents) into the human body is quite an attractive rationale for modulating the host immune system and gut microbiota, enhancing the symbiotic homeostasis of the super-organism, and providing protection against pathogens ( e.g. , meningitic E. coli ). Lactic acid bacteria (LAB), nonpathogenic antibiotic-resistant ascospore yeasts, and principally Saccharomyces boulardii are the most commonly used probiotics. A wide range of product types such as lyophilized form and fermented food products containing viable microorganisms with probiotic properties are commercially available  . In this study, the commercial probiotic agents used were Live Combined Bifidobacterium and Lactobacillus tablets containing original Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus . It is a dynamic combination of probiotic agents that is full of activity and colonization rates, which can quickly add to the intestine so that the number of intestinal probiotics could be restored to a healthy level.
Our previous studies have demonstrated that two subsequent steps, consisting of bacterial adhesion and invasion, are of great importance for pathogen access to the host cells. In order to cause meningitis, bacterial pathogens such as meningitic E. coli K1 must penetrate across the gut immune barrier and the BBB  . E. coli K1 binding to and penetration across the gut barrier is a prerequisite for bacteria entering into the blood circulation system (bacteremia) [6 , 28 , 44] . Neonatal bacterial meningitis usually develops as a result of bacteremia, which is one of the two key pathogenesis processes for meningitic pathogens to penetrate the brain. Bacterial traversal of the BBB due to high-level bacteremia is the second essential step for the evolution of bacterial meningitis  . To investigate the prophylactic efficacy of probiotics in neonatal bacterial bacteremia and meningitis, we had previously examined the suppressive effect of Lactobacillus rhamnosus GG (LGG) on meningitic E. coli K1 infection by using both the in vitro (Caco-2, a human epithelial cell culture model) and in vivo (the neonatal rat model of E. coli bacteremia and meningitis) models  . LGG could block E. coli K1 adhesion, invasion, and transcytosis in a dosedependent manner in Caco-2. The levels of pathogen colonization, E. coli bacteremia, and meningitis were significantly decreased in the LGG-treated neonatal rats when compared with those in the control groups. The underlying mechanism remains unclear, however, how probiotics enhance the host defense against bacterial pathogens.
The intestinal epithelial cells are involved in the formation of a physicochemical barrier and possess innate protective strategies against pathogenic challenge  . Others have indicated that intestinal tract mucins, located on the surface of the intestinal epithelium, acted as a protective layer against microbial damage and inhibit bacterial translocation [21 , 24 , 41] . MUC2, the main secreted mucin in the small and large intestines, is considered to be the major structural gel-forming component of the mucus gel [16 , 23 , 41] . Studies indicated that MUC2 mucin is an important regulatory factor in the gut immune systems, and selected lactobacilli may be able to induce the upregulation of MUC2 gene expression  . It has been shown that mucin-mediated gut barrier functions and beneficial effects of probiotics are enhanced by S-adenosyl-L-methionine, whose production is up-regulated by 5-aza-2-deoxycytidine (5-aza-CdR) [5 , 14 , 43] . As mentioned before, enteric bacterial pathogens crossing the gut barrier is the first critical step to cause bacteremia and then meningitis. In this report, the in vitro and in vivo model systems of the gut barrier and the BBB were used to examine whether MUC2, a protective layer against microbial damage, could play an active role in the host defense against bacterial translocation. Indeed, our studies on down- and up-regulation of MUC2 expression have shown that MUC2 is required for probiotics to efficiently block E. coli -induced bacteremia and meningitic infections.
Live Combined Bifidobacterium and Lactobacillus tablets (Trade Name: Golden Bifid; 0.5 g/tablet, containing no less than fifty million living Bifidobacterium , five million living Lactobacillus bulgaricus and Streptococcus thermophilus ) were generously provided by Inner Mongolia ShuangQi Pharmaceutical Co. Ltd. The three active components of the tablets were produced according to authorized manufacturing production and identifying procedures. The probiotic agents were grown in Lactobacilli MRS Broth (MRS Hardy Diagnostics, Santa Maria, CA, USA) at 37℃ for 24 h without agitation. To examine the molecular identities of the commercial probiotic agents containing Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus , PCR analysis and DNA sequencing were performed [48 , 52] . The bacteria genomic DNAs were extracted with a bacteria genomic DNA extraction kit (TIANGEN, China) according to the manufacturer's instructions. The 16S RNA gene sequences of Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus were obtained from GenBank, and the corresponding PCR primers of the specific 16S RNA gene were designed using Primer 5.0 software (Table S1). PCR amplification was performed with a Biometra PCR amplification system (UNOII-Themoblockt, Germany) and DNA fragments were amplified as follows: initial heating at 94℃ for 2 min, followed by 30 cycles of denaturation at 94℃ for 20 sec, annealing at 55℃ for 40 sec, extension at 72℃ for 30 sec, and a 7 min final extension step at 72℃. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence. In this analysis, mean values of three separate experiments were calculated Results showed that the PCR products were of the expected length (Fig. S1).
E. coli K1 strain E44, a clinic E. coli RS218 (O18:K1:H7) isolate from the CSF of a newborn infant with meningitis, is a rifampicinresistant strain. E44 was grown in brain heart infusion (BHI) broth with rifampicin (50 μg/ml) at 37℃ overnight without shaking [26 , 55] .
Human colonic carcinoma cell line Caco-2 (ATCC, Rockville, MD, USA) was maintained in 25 mmol/l glucose-Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mmol/l glutamine, 1% nonessential amino acids, and 1% penicillin/streptomycin at 37℃ in a humidified atmosphere with 5% CO 2 . Cells were used within 14 days of seeding or within 5 days of confluence  .
One day before treatment, Caco-2 cells were seeded in a 24-well plate (10 5 cells/well) and grown to confluence. The cell monolayers were treated with 5 × 10 -6 mol/l 5-Aza-CdR (freshly prepared) respectively for 72 h [22 , 42 , 49] . The medium was changed with the same concentration every 24 h. The cells without 5-Aza-CdR treatment were the control group, which was treated with an equal volume of medium instead of 5-Aza-CdR culture medium. The wells were washed twice with sterile PBS and 0.5 ml/well experimental media without antibiotics was added before bacterial treatment/infection.
The probiotic agents-induced blocking effects on meningitic pathogens were examined in the neonatal rat model of E. coli K1 strain E44 meningitis. All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University (SMU). All protocols were submitted and validated by the Animal Care Ethics Committee of SMU (No. 2012–055). A total of 20 pathogen-free Sprague Dawley rats (5 pups/group) were pooled together and randomly distributed into four groups (I: probiotics; II: probiotics + E44; III: E44; and IV: PBS). The 2-day-old pups were given probiotic agents (10 10 CFU/pup/day) by feeding using an FB Multiflex tip for 3 days. Control group and E44 group rats were fed PBS only  . At 5 days old, pups in the probiotics + E44 group and E44 group received E44 (10 9 CFU/pup), and pups in the probiotics group and control group received the same volume of PBS only. Intestine, blood, and CSF samples were plated onto LB agar plates with rifampin (50 μg/ml) after E44 inoculation for 48 h. Intestinal colonization, bacteremia, and meningitis were defined as a positive intestine culture, a positive blood culture, and a positive CSF culture, respectively. Colon tissue samples were washed with PBS and stored in RNAlater Storage Solution (Sigma) for RT-qPCR.
In order to determine the role of MUC2 in the probiotic agentsinduced preventing and blocking effects on meningitic E. coli ., total RNA in colon tissues and Caco-2 cells were extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer's recommendations, and then reverse transcribed in a 20 μl reaction system using the RT kit (Takara, Japan) according to the manufacturer's protocol. For RT-qPCR experiments, the forward and reverse primers corresponding to MUC2 and β-actin were designed using Primer 5.0 software ( Table 1 ). This was performed with an Mx3000P Real-Time PCR system and SYBR Premix Ex Taq II (Perfect Real Time) Kit (Takara, Japan) under the following PCR conditions: 95℃ for 30 sec; 40 cycles od 95℃ for 5 sec, 60℃ for 30 sec, and 72℃ for 5 min. The MUC2 mRNA expression levels relative to β-actin were calculated by the 2 -ΔΔCt method  . In this study, data from three separate experiments were averaged.
Target gene primers corresponding to MUC2 and β-actin.
We used the short hairpin RNA (shRNA) from the pGPU6/GFP/Neo vector containing the human U6 promoter (GenePharma, Shanghai, China) to knock down the MUC2 gene in Caco-2 cells. The scrambled shRNA was defined as a negative control (marked as "NC" in the figure). The recombinant constructs were verified through analyzing the fragments generated from digestion with Bam HI and by DNA sequencing. The interference efficiency was analyzed by RT-qPCR.
Caco-2 cells were seeded in a 12-well plate and grown to 85%-90% confluence. Cells were incubated in the transfection mixtures of shRNA plasmids and Lipofectamine 2000 reagent (Invitrogen, USA) for 48 h according to the manufacturer's protocol. Transfected cells were observed via fluorescence microscopy to estimate the transfection rate. Cells transfected with siRNA oligos containing no homology with any human gene were used as the negative control  .
To detect MUC2 protein expression in Caco-2 cells, western blot analysis was performed. For the preparation of lysates, the cells were washed with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS) supplemented with protease inhibitors. Cells were scraped and centrifuged at 4℃ for 15 min. The supernatant was obtained, and the protein concentration was determined using the Bradford method (Bio-Rad). Samples were then prepared by mixing with 4× SDS sample buffer and boiling for 5 min, and then subjected to 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA)  . The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibody (anti-MUC2 antibody purchased from Abcam) at 1:500 under constant agitation overnight at 4℃. After washing with PBST, the membranes were incubated with HRP-conjugated secondary antibody (anti-rabbit, 1:5,000) for 1h at room temperature under constant agitation. Proteins were developed using an enhanced chemiluminescence system (ECL; Amersham Biosciences, USA).
The bacterial adhesion and invasion assays were performed as described previously to assess exclusion of E44 strain by probiotics agents [6 , 28] . For the competition assay, Caco-2 cells were seeded in a 24-well plate and then pre-incubated with 1 × 10 8 CFU of probiotic agents and 1 × 10 7 CFU of E44 in the EM (experimental medium) at 37℃ for 1.5 h to allow adhesion and invasion to occur.
For adhesion assays, the cells were washed three times with PBS to remove unattached bacteria. The bacteria bound to intestinal mucus were released and lysed with 200 μl of 0.5% Triton X-100 for 8 min, followed immediately by the addition of 150 μl of sterile water. The lysate samples were diluted with a double dilution method and cultured on LB agar plates containing rifampin to determine the total number of colonies (CFU/ml) recovered from the lysed cells. Each experiment was repeated for three times. Results were indicated as relative adhesion (adhesion% as compared with the adhesion of the control E44 group).
For invasion assays, the cells were washed three times with PBS, and incubated with EM containing gentamicin (100 μg/ml) for 1 h at 37℃ to eliminate the extracellular bacteria for the determination of the number of intracellular bacteria. Each experiment was repeated for three times. Results were indicated as relative adhesion (adhesion% as compared with the adhesion of the control E44 group).
Data analysis was performed as described previously  . ANOVA and covariates followed by a Student-Newmann-Keuls test for a multiple comparison test were performed to determine the significant difference between the control and experimental groups. P < 0.05 was considered as statistically significant.
The intestinal mucus layer and gut microbiota are strongly intertwined and this would be conducive to the maintenance of the intestinal epithelial barrier and immune intestinal homeostasis  . Our in vivo studies showed that probiotic agents could block E. coli -caused intestinal colonization, bacteremia, and meningitis in a rat model of NSM ( Figs. 1 A, 1 B, and 1 C). We tested the effect of the probiotic agents and E44 on baseline mucus expression in rats and assessed the regulation of the MUC2 gene in the presence of probiotics and E44 bacteria. MUC2 expression was based upon the relative expression as compared with β-actin. As had been expected, the mRNA levels of MUC2 mucin were up-regulated in the probiotics group. In rats with E44, MUC2 gene expression was lower than in the control group. Conversely, no changes in MUC2 were detected in rats fed intragastrically by probiotic agents and E44 ( Fig. 1 D). These results indicated that the MUC2 gene expression in rat colon could be selectively stimulated by probiotic agents, and these rats may up-regulate MUC2 mucin synthesis as a defense mechanism.
Blocking effects of probiotic agents on intestinal colonization, bacteremia, meningitis, and MUC2 expression in rat colon tissues after receiving probiotics and E44.	A total of 20 rat pups were randomly divided into four groups; (I: probiotics group; II: probiotics + E44 group; III: E44 group, IV: control (PBS) group). For groups I and II, probiotic agents (1010 CFU/pup/day) were given orally to 2-day-old rats for 3 days before E44 infection (109 CFU/pup) for 2 days. At 7 days old, the intestine, blood, and CSF samples were obtained and used for bacterial culture in LB agar plate with rifampin (50 μg/ml) for displaying the intestinal colonization (A), bacteremia (B), and meningitis (C), respectively. (D) The total RNAs of intestine samples were isolated and the expression of MUC2 was analyzed with RT-qPCR. In contrast to the control group, the mRNA levels of MUC2 were up-regulated in the probiotic agent-treated group. MUC2 expression was based on the relative value as compared with β-actin. **P < 0.01 vs. control group.
To gain further insight into the particular function of MUC2 in Caco-2 cells, we employed an RNAi module to selectively down-regulate MUC2 expression. A total of four plasmids were constructed and named as pGPU6/GFP/Neo/MUC2-1, pGPU6/GFP/Neo/MUC2-2, pGPU6/GFP/Neo/MUC2-3, and pGPU6/GFP/Neo/MUC2-4, respectively. Proper construction of the plasmid was confirmed by cutting with restriction enzymes. Positive clones were verified by DNA sequencing (data not shown). The state of transfected cells was evaluated by fluorescence microscopy, which showed the transfection efficiency was approximately 90%. The mRNA levels of MUC2 were tested by RT-qPCR. As indicated in Fig. 2 A, mRNA expression of MUC2 in Caco-2 cells transfected with pGPU6/GFP/Neo/MUC2-1, pGPU6/GFP/Neo/MUC2-2, pGPU6/GFP/Neo/MUC2-3, and pGPU6/GFP/Neo/MUC2-4 were reduced by 78%, 41%, -14%, and 62%, respectively, in comparison with the control cells. To determine the inhibition efficiency of MUC2 protein expression, western blot analysis was done. Similar to the results of RT-qPCR analysis, pGPU6/GFP/Neo/MUC2-1 exerted the strongest effect in suppressing MUC2 expression ( Fig. 2 B). Values for the fold-increase are presented as mean ± SEM of three independent experiments as quantitated by densitometry ( Fig. 2 C). Therefore, we selected pGPU6/GFP/Neo/MUC2-1 for the further experiments.
MUC2 knockdown efficiency in Caco-2 cells.	Caco-2 cells were cultured in 12-well plates. The cells reaching 85-90% confluence were incubated with both mixtures of shRNA plasmids and Lipofectamine 2000 reagent for 48 h. The transfection rate was estimated with a fluorescence microscope and MUC2 knockdown efficiency was detected by RT-qPCR (A) and western blotting (B-C). β-Actin was used as a loading control, and the expression level of MUC2 mRNA was normalized with β-actin (n = 3, means ± SD). The densitometric analysis of MUC2/β-actin ratio was performed using the Gel-Pro Analyzer 4.0 software.
We hypothesized that probiotic agents could suppress bacterial penetration across the gut barrier and bacteremia/meningitis through enhancing host MUC2 defence functions. In order to investigate the role of MUC2 in probioticsmediated blocking effects on meningitic E. coli -induced pathogenicity, the competitive exclusion assay was perform. We examined the ability of probiotic agents to intervene in the adhesion and invasion of meningitic E. coli E44 to Caco-2 cells. As shown in ( Fig. 3 ), probiotic agents were able to emulously suppress E44 invasion and adhesion in Caco-2 cells (* p < 0.05, ** p < 0.01). In contrast, after MUC2 silencing, the inhibition by probiotics was significantly lower than the untreated group. The relative adhesion rate of E44 was 63.64% ( Fig. 3 A), and the relative invasion rate of E44 was 70.33% ( Fig. 3 B). The invasion and adhesion rates of E44 at the zero concentration of probiotic agents were assigned as 100% and the effects of probiotic agent pre-incubation were compared with this control level.
MUC2 is required for probiotic agents-induced blocking effects on E44 adhesion and invasion.	Caco-2 cells were cultured into a 24-well plate and grown as a monolayer, and 1 × 108 CFU of probiotics mixtures and 1 × 107 CFU of E44 were added to incubate for 1.5 h. Adhesion (A) and invasion (B) assays were performed as described in the Materials and Methods section. The results are presented as relative adhesion% and invasion% compared with that of the control group without probiotics. *P < 0.05, **P < 0.01.
In order to investigate whether 5-Aza-CdR could enhance the biological activity of probiotics through up-regulation of MUC2, which may be activated by SAMe, we first analyzed and compared the changes of MUC2 expression before and after adding with 5-Aza-CdR. We used 5-Aza-CdR to selectively up-regulate MUC2 expression in Caco-2 cells. RT-qPCR analysis showed that the promoter region of MUC2 exhibited a demethylation state and induced MUC2 re-expression. In the 5-Aza-CdR and probiotics group, MUC2 expression was significantly up-regulated. However, MUC2 expression was much lower in the E44 group (** p < 0.01, Fig. 4 ).
Comparative analysis of the effect of 5-Aza-CdR and probiotic agents on MUC2 expression in Caco-2 cells.	Caco-2 cells were seeded at a density of 105 cells/well in a 24-well plate to become confluent, 5 × 10-6 mol/l 5-Aza-CdR (freshly prepared) was stimulated with the cells for 72 h, and the total mRNA was then collected. RT-qPCR was carried out and the expression of MUC2 mRNA was normalized with β-actin (n = 3, means ± SD). **P < 0.01 compared with control group.
Next, we tried to test if probiotic effects could be enhanced through 5-Aza-CdR-mediated demethylation of the MUC2 gene in Caco-2 cells. The ability of 5-Aza-CdR and probiotic agents to interfere with the pathogen E44 adhesion and invasion in Caco-2 cells was examined by competitive exclusion assays after treating with 5-Aza-CdR for 72 h. As shown in Fig. 5 , 5-Aza-CdR was able to strongly inhibit E44 penetration of Caco2 cells. The relative adhesion rate of E44 was 10.33% ( Fig. 5 A) and the relative invasion rate of E44 was 29.45% ( Fig. 5 B). The result of the probiotics group was similar to 5-Aza-CdR treatment (** P < 0.01). The adhesion and invasion rates of E44 at the zero concentration of probiotics and 5-Aza-CdR were assigned as 100% and the effects of 5-Aza-CdR and probiotics preincubation were compared with this control level.
Blockage of E44 penetration across Caco-2 cells by 5-Aza-CdR and probiotic agents.	Caco-2 cells seeded in 24-well plates were incubated with 1 × 108 CFU probiotic agents and 1 × 107 CFU bacteria (E44) for 1.5 h, after treating with 5-Aza-CdR (5 × 10-6 mol/l) for 72 h. Adhesion (A) and invasion (B) assays were performed as described in the Materials and Methods section. The results were presented as relative adhesion% or invasion% compared with that of the control group without probiotic agents or 5-Aza-CdR. **P < 0.01.
The use of probiotic microorganisms for the prevention and treatment of disease has become increasingly popular over the past few decades because of increased scientific studies that demonstrate the favorable effects of probiotics on human health  . The most frequently used probiotic agents is LAB, such as Lactobacillus rhamnosus GG, which has been examined for its efficacy in prevention of neonatal sepsis in both animal models and humans caused by enteric bacterial pathogens [2 , 26 , 39 , 40] . Probiotic LGG supplement in neonates, including very low birth weight newborns, are confirmed to be microbiologically safe as well as clinically well tolerated. Moreover, Lactobacillus and Bifidobacterium are the most commonly used probiotics. In our studies, the probiotic we used was the commercially Live Combined Bifidobacterium and Lactobacillus Tablets, which could inhibit E. coli K1 strain E44 intrusion into the intestinal epithelial cells in vitro and be able to reduce the incidence of bacteremia and meningitis in vivo . It was indicated that the significant reduction of pathogens across the gut barrier results in the release or elimination of bacteria access into the bloodstream, which eventually blocks pathogens from crossing the BBB to cause meningitis.
The mucin-containing cells in the surface of the gastrointestinal tract provide a physical protection and an interface between the internal environment and the dynamic external challenge from food-derived antigens and microbes  . Pathogens and benign microbes are discriminated by the gut mucosal immune system, which induces protective immunity without disrupting the integrity of the gut mucosa. Some studies have shown that the intestinal mucin gene MUC2 was constitutively expressed by the confluent human intestinal epithelial (Caco-2) cells [16 , 34 , 38] . In this study, we investigated the role of MUC2 in probiotics-mediated blocking effects on meningitic E. coli -induced pathogenicity. We hypothesized that probiotic agents could suppress bacterial penetration across the gut barrier and bacteremia/meningitis through enhancing host MUC2 defence functions. Probiotic agents induce up-regulation of epithelial MUC2, which in turn inhibits bacterial invasion and bacteremia/meningitis. We found that Caco-2 cells treatment with probiotic agents could be induced to present higher expression of MUC2. We previously demonstrated that Lactobacillus rhamnosus GG could inhibit pathogen penetration in vitro and in vivo  . In this report, we further demonstrated that probiotic agents could block bacterial invasion, bacteremia, and meningitis through up-regulation of MUC2 production.
Numerous factors, such as the imbalance of nutrition, antibiotics use, immunosuppressive therapy, and other means of treatment, might induce alteration both in the composition and effects of the human normal microbiota that may influence the efficacy of probiotics  . Previous studies demonstrated that S -adenosyl-L-methionine is a key nutritional factor for mucosal foraging and fitness of probiotic microorganisms and the prominent human gut symbiont, Bacteroides thetaiotaomicron [5 , 7 , 43] . Most interestingly, a synergistic anti-breast cancer effect can be achieved by a combined treatment with the methyl donor SAMe and the DNA methylation inhibitor 5-Aza-CdR  . 5-Aza-CdR could increase production of SAMe and sialyl Lewis X on MUC1 by activation of the gene coding for β-galactoside, α2,3-sialyltransferase 6, via inhibition of DNA methylation [8 , 35] . Based on these findings, we hypothesized that 5-Aza-CdR could enhance the biological activity of probiotics through up-regulation of MUC2, which may be activated by SAMe. To test this hypothesis, we tried to investigate if probiotic effects could be enhanced by 5-Aza-CdR-mediated demethylation of the MUC2 gene in Caco-2 cells. By analyzing and comparing the changes in MUC2 expression before and after treatment with 5-Aza-CdR, we speculated that MUC2 mRNA expression is associated with the methylation of the 5'CpG island of the MUC2 promoter. The changes in methylation of the MUC2 gene may be highly correlated with the inhibition of MUC2 transcription. Research has shown that 5-Aza-CdR could reverse CpG island methylation and restore the expression of multiple tumor suppressor genes [4 , 50] , which is consistent with our present study. This provides us with a new approach to study the relationship among probiotics, pathogens, MUC2, fitness factors ( e.g. , SAMe), and the specific mechanism of probiotics and its role in pathogensis. The underlying mechanisms responsible for up-regulation of MUC2 and SAMe by 5-Aza-CdR remain to be defined.
In conclusion, probiotic agents represent a promising host-directed antimicrobial agent that can be developed as a novel therapeutic intervention through enhancing host defence against infections, neonatal sepsis, and bacterial meningitis. Additionally, our data suggest that probiotic agents can efficiently block meningitic E. coli -induced pathogenicity in a manner dependent on MUC2.
This study was partially supported by the National Natural Science Foundation of China (No. 81370740 to S.H. Huang), Public Health Service grants R21NS083967 (S.H. Huang), the Grant from School of Public Health and Tropical Medicine of Southern Medical University, China (No. GW201248), and Guangdong Province Undergraduate Innovative Experiment Program (No. 1212112016 & No.1212112017).
 JY Yu , XL He , S Puthiyakunnon , L Peng , Y Li , LS Wu , WL Peng , Y Zhang , J Gao , YY Zhang , S Boddu , M Long , H Cao , and SH Huang , "Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced PathogenicitiesS", Journal of Microbiology and Biotechnology, vol. 10, no. 10, Oct 2015.


The best part of my day was having a striper hit the top water lure, sending in the air, then coming back and hitting it again with a tail slap, when I realized, it was 3 Stripers fighting over it.
When Finally it Happened, I Had a Double on, and it was 2 Good size Keepers, they were rolling and fighting when one popped off.
But the one I got in the boat was the 32 1/2″ fatty. I ended up with 10 Stripers, ranging from 24″ to the 32 1/2″.
Plus, I was soooooooooo tired from standing and fishing all morning.
That W500 is a work out machine, just as much as she is a FISHING MACHINE!!!!!!!
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And surely a major cause is government privileges for private credit creation, i.e. for "the banks" since the rich are, by definition, the most so-called "credit worthy" of what is then, in essence, the public's credit but for private gain.
And no, those privileges are not necessary in order to have low interest rates.


a) the Tappiya waterfall after Batad. With a phenomenal 70 meter fall in rainy season and a naturally formed pool at its bottom suitable for a cooling swim (dress conservatively), Tappiya waterfall is a unique location in the area.
a) Witness the historic, old Spanish towns in Vigan and Laoag.
b) Visit Lingayen Gulf and go half day island hopping amongst the Hundred islands.
d) Go trek Mount Pinatubo volcano.
e) Visit Subic, Philippines former US Naval Base and now a freeport zone.
f) Visit Baguio the summer capital of North Luzon.
The Mayoyao Rice Terraces are one of the five clusters included in the UNESCO World Heritage inscription Rice Terraces of the Philippines Cordilleras. Its known for the Apfo' or Burial Tombs which are large stone, dome-shaped mausoleums found only in Mayoyao. Bones of Mayoyao's ancient warriors and elite citizens were buried in these tombs. During the World War II Mayoyao was one of the places where a battle was fought between the Filipino/American forces against the Japanese Imperial forces which led here to the capture of General Yamashita and eventually his surrender at Kiangan. This tour offers an extra ordinary experience in this beautiful town where unexplored nature still bounds.
10:00 am – Our driver/guide will pick you up at Cauayan airport and then we drive via Alicia to Mayoyao in 2-3 hours along the very scenic roads with deep cliffs along the side when approaching our destination. Underway there are a few scenic lookout points before arriving around 1-2 pm in Mayoyao where we stay overnight in Helen's place. After Lunch we will tour the area and enjoy the Mayoyao rice terraces and have a panoramic view of the villages. The Apfo' or are domed-shaped Burial Tombs, found only in Mayoyao and use to house the bodies of the town's ancient warrior and elite. General Tomoyuki Yamashita of the Japanese Imperial army in the Philippines was captured here on August 1945 and brought to Kiangan where he surrendered before he was taken to Baguio City where is formal surrender was staged, tried for war crimes and hanged in Los Baños in 1946.
08:00 am – We have an early breakfast and then we depart for Batad which is a 2 hour drive. We will make (photo) stop at the Bangaan rice terraces. Our driver will arrange a local guide and drop you at the Batad saddle and the rest of the distance is on foot using a mountain trail, takes only a 30 minutes hike to Batad viewpoint (*). The trip to Barangay Batad in Banaue, to witness the amphitheater shaped terraces located at 3600 ft. At Batad viewpoint you can take your lunch while enjoying the scenery.
After breakfast in the hotel, we start the day with a 2.5 hours ride (one way) to picturesque Sagada with its ancient burial caves and mummified bodies at Sugong & Echo Valley.
Under guidance of a local English speaking guide you will glimpse Lumiang Burial Cave which houses more than 100 coffins are stacked in the entrance. The oldest around 500 years old. Many of them have images of lizards carved on them which are symbol of fertility and long life. Visit Bontoc's Tribal Museum with artifacts and unique pictures from the mountain tribes.
We then enter the impressive Sumaguing Cave, known for it's rock formation and impressive chambers. In the afternoon (optional and when time allows) we visit the Bomod-ok waterfalls. The track to the waterfalls takes a total of 3 hours. Overnight in Masferre guesthouse.
Leave at 07:00 am after breakfast going to Baguio in 5 hours where you may take your lunch at Camp John Hay Manor Hotel. After lunch we proceed to Manila where we will arrive at approximately 18:30 pm depending on the weather and traffic conditions.
-Roundtrip transportation by airconditioned vehicle Cauayan- Mayoyao-Manila (FREE WIFI onboard for first 3 bookings) with English speaking driver.


text:
package org.jpublish.module.wink.examples;

import org.apache.commons.logging.Log;
import org.apache.commons.logging.LogFactory;
import org.apache.wink.common.internal.utils.MediaTypeUtils;
import org.apache.wink.common.model.multipart.InMultiPart;
import org.apache.wink.common.model.multipart.InPart;
import org.apache.wink.common.model.synd.SyndEntry;
import org.apache.wink.common.model.synd.SyndText;
import org.jpublish.util.FileCopyUtils;

import javax.ws.rs.*;
import javax.ws.rs.core.MediaType;
import javax.ws.rs.core.MultivaluedMap;
import java.io.File;
import java.io.FileOutputStream;
import java.io.IOException;
import java.io.InputStream;
import java.util.Date;
import java.util.regex.Matcher;
import java.util.regex.Pattern;

/**
 * Simple example - Hello World!
 *

 * The SDK dispatches HTTP requests for URI
 * http://[host]:[port]/HelloWorld/rest/world, where
 * HelloWorld is the context root, to this class. A simple Atom
 * entry is returned in HTTP response.
 *

 * The service document is available at URI
 * http://[host]:[port]/HelloWorld/rest but it is empty because
 * this simple demo doesn't contain any collection of resources.
 *

 * This resource must be registered within a JAX-RS application, this example
 * uses the default usage of application /WEB-INF/application
 */

//

/**
 * if you add this: @Path("/wink/world")
 * and ask:
 * $ curl -H "Accept:application/atom+xml; charset=utf-8" "http://localhost:8080/wink/world"
 * you get this:
 * <?xml version="1.0" encoding="utf-8" standalone="yes"?>
 * <entry xmlns:ns3="http://www.w3.org/1999/xhtml" xmlns:ns2="http://a9.com/-/spec/opensearch/1.1/" xmlns="http://www.w3.org/2005/Atom">
 * <id>helloworld:1</id>
 * <updated>2010-04-12T16:50:31.958-04:00</updated>
 * <title type="text">Hello World!</title>
 * </entry>
 */
@Path("/wink/world")
public class HelloWorld {
    protected static final Log log = LogFactory.getLog(HelloWorld.class);
    public static final String ID = "helloworld:1";

    /**
     * This method is called by the SDK for HTTP GET method requests where the
     * Accept header allows the Atom media type application/atom+xml. A
     * SyndEntry is created with basic information. Serialization of the
     * SyndEntry to Atom entry is performed by the SDK automatically. The
     * default status code of 200 (OK) is returned in the response.
     *
     * @return SyndEntry of the requested resource
     */
    @GET
    @Produces(MediaType.APPLICATION_ATOM_XML)
    public SyndEntry getGreeting() {
        // create and return a syndication entry with a "Hello World!" title,
        // some ID and the current time.
        SyndEntry synd = new SyndEntry(new SyndText("Hello World!"), ID, new Date());
        return synd;
    }

    @GET
    @Produces(MediaType.TEXT_HTML)
    public String getGreetingHtml() {
        return "Hello World!";
    }

    /**
     * demonstrating a multipart transfer (file upload) with Wink. Expects a file name as a
     * path parameter. The file name will be used to create a temporary file. Example of usage:
     *
     * curl -H "Accept:text/plain" -F "name=@text.txt"  http://localhost:8080/wink/world/file/file_out
     *
     * @param inMultiPart the multipart form data
     * @return a string confirming the file uploaded and its size
     * @throws IOException
     */

    @Path("file/{fileName}")
    @POST
    @Produces(MediaType.TEXT_PLAIN)
    @Consumes(MediaTypeUtils.MULTIPART_FORM_DATA)
    public String uploadFiles(InMultiPart inMultiPart, @PathParam("fileName") String fileName) throws IOException {
        StringBuilder sb = new StringBuilder();

        while (inMultiPart.hasNext()) {
            File f = File.createTempFile(fileName, ".tmp");
            FileOutputStream fos = new FileOutputStream(f);

            InPart part = inMultiPart.next();
            MultivaluedMap<String, String> headers = part.getHeaders();

            String CDHeader = headers.getFirst("Content-Disposition");
            InputStream is = part.getInputStream();

            int size = FileCopyUtils.copy(is, fos);

            String fn = "";
            Pattern p = Pattern.compile("filename=\".*\"");
            Matcher m = p.matcher(CDHeader);

            if (m.find()) {
                fn = m.group();
            }

            //String filename = CDHeader.
            sb.append("uploaded a file, ").append(fn).append(", size = ").append(size).append(" bytes\n");
            log.info(String.format("%s, created", f.getAbsolutePath()));
        }
        return sb.toString();
    }

}

redpajama_set_name: RedPajamaGithub


text:
Molina wins, Wittmann bat to Mortara by the minimum
October 15, 2016 by jason
Miguel Molina has won his third victory in the DTM
the final round of the championship in Hockenheim. The Spanish rider, oblivious to the great
battle for the drivers title, signed a perfect outlet that allowed him to
get the first position and dominate the race from the first corner until
the checkered flag.
behind and team orders by Marco Wittmann
I got the second position
, which eventually served to overcome Edo
Mortara. The Italian downplayed damage with a great comeback, although their
title options have been reduced still more.
In a perfect output, Miguel Molina was placed leader after overtaking the poleman António Félix Da Costa. Third, for its part, and without being involved in the battle between the two is positioning itself Marco Wittmann, an attitude of cautious and logical to be escorted by Tom Blomqvist. In fact, Wittmann came out winning because his rival for the title, Edo Mortara is the I was stiff with Timo Glock. The German made a perfect team work to bring the Italian to the eighth position with a maneuver as a minimum set. Behind, Dani Juncadella won five positions in the output to be seventeenth.
After a first offense of António Félix Da Costa, Miguel Molina got a space of about three seconds on the Portuguese, which led to BMW Motorsport to run the first movements to secure the drivers title. António Félix Da Costa is allowed to pass by Marco Wittmann, leaving the German second and as a champion virtual of the DTM, every time that the role of judge that I could have a Mercedes left in anything with six of its eight cars in the last six positions. Felix Rosenqvist put the icing on the drama of the signing of Stuttgart abandoning half of the race.
despite their determined defence, Edo Mortara was stealing the wallet to Timo Glock, which in turn allowed the Italian to hunt Augusto Farfus and overtake the brazilian. Placed in the sixth position, Mortara is put into chase mode to give chase to Mike Rockenfeller, knowing that their potential partner in the Team Abt Sportsline, he was going to cede the position. Complied with the procedure, Mortara are not used to take or a spin on take off the middle Tom Blomqvist to climb to the fourth position, thereby reducing the points lost compared to Wittmann and taking a step forward to achieving the title of equipment to the Audi.
The overtaking of Mike Rockenfeller and René Rast on Tom Blomqvist also served to improve the result of Audi in their fight with BMW for the title of brands, although all the attention came to fall on the front already that Edo Mortara signed a large 'cavalcade' to be placed in the wake of António Félix Da Costa. The Italian thought for a second and with an overtaking to the limit Mortara got the third place of the podium with two rounds played. Insufficient in any case to be able to overcome Wittmann.
all in all, Miguel Molina signed his third victory in the DTM in a race shaped in a large output. Second crossed the finish line Marco Wittmann, while Edo Mortara finished third. With this result, Wittmann gets three extra points in the battle for the title and will face the last race of the season with 17 units of advantage on Mortara. Off the podium and fight for the title, António Félix Da Costa was fourth, followed by German Mike Rockenfeller, who confirmed the bet of Audi giving a few valuable points to the Team Abt Sportsline in relation to the eighth joined by Timo Glock.
Classification first career DTM 2016 at Hockenheim II
Position Pilot Vehicle Time / Distance
1 Miguel Molina Audi RS 5 DTM 43:02:346
2nd Marco Wittmann BMW M4 DTM +1.661
3rd Edo Mortara Audi RS 5 DTM +2.805
4th António Félix Da Costa BMW M4 DTM +4.870
5th Mike Rockenfeller Audi RS 5 DTM +6.064
6th René Rast Audi RS 5 DTM +7.992
7th Timo Glock BMW M4 DTM +11.858
8th Jamie Green Audi RS 5 DTM +15.970
9th Tom Blomqvist BMW M4 DTM +17.180
10th Paul Di Resta Mercedes AMG C 63 DTM +19.048
Atieva Atvus: it filters the first image of the future rival to the Tesla Model S
SEAT sell the Mii by Mango in France with Amazon

redpajama_set_name: RedPajamaCommonCrawl


After the Golden Globes I'm feeling a little extra fashion-y today. In thinking about it, my favorite dress was Lanvin that Natalie Portman wore. So pretty, elegant, feminine, dreamy.
That reminded me of how smitten I am with the Lanvin petit collection for 2-to-8-year-olds. This past summer, Lanvin's Alber Elbaz reawakened the house's childrenswear roots, unveiling his Lanvin Petite 25-piece collection in New York. The line features separates, dresses and a few select accessories. Complete and total Silver Lining.
Now, I really don't think that even if I had all of the money in the world that I could rationalize spending over $1000 on a dress for Finally Five. Come to think of it, there's not a chance in H**L, especially if I want to stay married to the HOTY. However, a girl can certainly dream, right?!?
That Alber Ebaz cuts a dress like nobody's business – and I did not know about the children's clothes. A revelation. Thanks as always for enlightening us.
Right?!? I love any and all Lanvin! Thank you, Frances!
Omg, how can you resist?
By looking at the price tag.
Absolutely adorable coats! Wouldn't have let my kids near these clothes even if we could have afforded it….. too many tempting mud puddles, snowdrifts & grassy playgrounds to be explored at that age!! ha ha ha…. Thanks for the peak!!
I couldn't agree more, Kim. Super fun to look at though! Thanks, as always.
Thanks for sharing these adorable fashions for the FF set. Your post made me laugh out loud! Big hug to all.


Tues 19 June: 19h00-22h00. Graduates meet socially and bring their friends.
Often, after the More To Life Weekend, we offer six evening sessions of three hours each to review all the processes that you learnt during the Course. These sessions are not always offered so please check with your Trainer on the last evening of the course.
Any outstanding balance is due by 13 June 2018.
To qualify for the Early registration fees, the tuition fee must be paid in full no later than 27 May 2018.
The discounted fee is available to retakes, full time students, senior citizens (65y and over), 2nd and following household members when registering together (e.g. couples).
If you cancel your booking within seven days of registering, with the exception of the week of the course itself, you may request a full refund, excluding the non-refundable deposit. After this time we will transfer your registration to the next available course that suits your availability.


Ocean dumping is currently used to dispose of industrial wastes, sewage sludge, garbage, construction debris, derelict vessels and dredged material. Most of these waste materials of our modern society are generated on land. Because of tidal activity, a high proportion of this waste remains within one kilometre of the coast, which contain the richest fishing grounds, tourist and other natural values. There are three principal modes of transferring these substances from the land into the sea: river discharge; atmospheric transport, followed by washout with rain; and coastal discharges through outfalls. Vessels contribute to a certain amount of pollution through discharge of sewage and garbage. Increasing quantities of material are being transported out to sea for dumping at designated dump sites; however, the largest amount of material will continue to be released into the sea through coastal outfalls.
The amount of solid materials entering the sea annually can be in the millions of tonnes. While this type of pollutant is largely cosmetic in character and affects mainly the amenities, there are other uses of the sea which are also affected. For example, large polypropylene or nylon ropes, floating just below the sea surface, can become entangled in the propellers of vessels, causing damage and possibly accidents at sea. Plastic sheets can also become entangled in ships' propellers, as well as clogging sea water intake systems for engine cooling and other purposes. Fishing gear may become fouled by netting and ropes, and on large solid objects deposited on the bottom of the ocean. From the ecological point of view, solids deposited on the bottom can adversely affect the benthic habitat. Plastic sheets can smother organisms beneath them, because of lack of oxygen replacement due to elimination or reduction of water exchange, and adversely affect the substrate for settlement of larvae. Plastic bags have been found on the heads of fur seals; plastic and rubber spherules have been found in the intestines of fish.
Of wastes dumped deliberately into the sea, as much as 80% consist of 'dredge spoils', the materials scraped from river and harbour bottoms to open channels and facilitate navigation. These materials are natural, for the most part, made up of sand, silt, clay and rock. They are usually barged to offshore areas and dumped, often grossly disrupting these undersea locations for a period of time. These 'spoils' often include a high proportion of deposited sewage sludge and accumulated industrial waste, with its potential toxicity.
Although about half of the oil transported in the world each year is carried by tankers, and tanker oil is dumped as part of routine operations, (such as illegal deballasting and tank washing, which flush oil into the sea), most of the oil in the sea comes from a combination of land-based industrial and municipal sources. As much as 350 million gallons (1.3 billion litres) of used automobile crankcase oil is dumped each year into water drainage systems that reach the sea. About 600,000 tonnes of oil run into the sea each year a result of petroleum carried into the atmosphere from poorly tuned automobile engines. Of the twenty largest metropolitan areas in the world, sixteen are coastal cities, or cities on rivers which empty relatively quickly into the ocean. About 30 billion gallons (113 billion litres) of industrial and municipal wastes are discharged into the coastal areas of the USA alone every year.
The chief danger of ocean sewage dumping lies in the spread of viral and bacterial diseases - directly to bathers, indirectly through fishery products. On the basis of the toxicity and the volume of many wastes entering the sea it seem likely that sea life could be seriously disrupted in some places more than others, but the threat is global.
The sea was the first sewage treatment plant. It has processed the organic waste and the dead husks of living things since life evolved there 3.5 billion years ago. The system thrives on this natural excrement. The animal life of the great depths would not survive without the nourishment of waste matter raining down from more populated surface waters. Gray whales contribute more faecal matter to the sea worldwide than the Los Angeles sewer system does; so do anchovies. There are also natural oil leaks in the sea. Seepage from sub-sea deposits wells up in many places around the world. Off the California coast, one natural seepage contributes about 660,000 tonnes of petroleum to the sea every year.
Because the sea and its organisms can handle this load of natural wastes quite well, the misconception has arisen that there is no limit to what the sea can dispose of in its self-purification systems. The human waste from a large city like Los Angeles is greatly concentrated in one location, not dispersed widely, and it is usually spiked with toxic substances that the sea may be unable to recycle naturally. There is natural seepage of petroleum into the sea from ocean floor deposits, but human-caused oil pollution is now about ten times that rising naturally from the ocean bottom. Perhaps the most pernicious misconception about the sanitation system of the ocean, however, is that coastal dumping is a negligible contribution to an environment so vast and deep, covering nearly three quarters of the planet's surface. The fact is that most of the sea's life is concentrated along the world's coastlines, where nutrient-rich currents pass across convenient hiding and attachment surfaces shallow enough to be touched by sunlight. Of the 140 million square miles (363 million square kilometres) of ocean surface, it is only the 14 million square miles (36 million square kilometres) near shore that contain the sea's most important living habitats. About 90% of the world's food fish spawn, mature, and are caught, in these coastal areas. Unfortunately, the land adjacent to these productive waters is and where human populations gather, where many industries locate, where the greatest mass of destructive waste matter is dumped.
Dumping was at sea is preferable to expanding disposal sites on land. Land dumping is more expensive, more difficult to gain approval of local authorities and more likely to meet with political opposition.


A Late News item on Page 2 of the June 16 issue contained inaccurate information from the National Automobile Dealers Association about changes in eligibility requirements for NADA's board of directors. Directors whose dealerships are bought out by public companies must resign as directors and a special election will be held to replace them. They will be eligible to run in the next regular election.
An article on Page 4 of the June 16 issue incorrectly described the formation of American Axle & Manufacturing Inc. The company was created from five plants that were spun off by General Motors' Saginaw Division, now Delphi Saginaw Steering Systems.


text:
Home > Universities in India and Abroad > Virginia Polytechnic Institute and State University
Virginia Polytechnic Institute and State University or Virginia Tech (VT) is a public land-grant university with over 125 campus buildings across the globe. Since the year 1872, it is considered one of the top-notch institutions in the world with the largest number of academic programs and full-time students.
The university has 65 undergraduate programs, 145 post graduate and doctoral programs, 1 professional course from the Virginia-Maryland Regional College of Veterinary Medicine to offer to the students. The Virginia Tech Carilion School of Medicine and Research Institute is the latest initiative taken by the university. Moreover, the institute offers online as well as distance learning programs named Virginia Tech Online (VTOnline) which includes 35 graduate degree and certificate programs. The undergraduate colleges, schools, and academic programs offered by each of them are listed below:
Colleges / Schools
AHNR Information Technology
Agricultural and Applied Economics
Agricultural and Extension Education
Animal & Poultry Sciences
Crop and Soil Environmental Sciences
Biological Systems Engineering
Human Nutrition, Foods and Exercise
Plant Pathology, Physiology, and Weed Science
School of Public and International Affairs
Government and International Affairs
Center for Public Administration and Policy
Urban Affairs and Planning
School of Visual Arts
Creative Technologies
Myers-Lawson School of Construction
Vecellio Construction Engineering and Management
College of Liberal Arts and Human Sciences
Learning Sciences and Technologies
Air Force ROTC (Military Aerospace Studies)
Apparel, Housing, and Resource Management
Army ROTC (Military Science)
Foreign Languages and Literatures — Classical Studies
Foreign Languages and Literatures — French and Francophone Studies
Foreign Languages and Literatures — Russian Section
Foreign Languages and Literatures — German Section
Foreign Languages and Literatures — Spanish Section
Navy ROTC (Naval Science)
Science & Technology in Society
Theatre and Cinema
Finance, Insurance, and Business Law
Aerospace and Ocean Engineering
Biomedical Engineering & Sciences, (VT-WFU School of)
Engineering Science and Mechanics
Mining and Minerals Engineering
College of Natural Resources and Environment
Fish and Wildlife Conservation
Sustainable Biomaterials
Forest Resources and Environmental Conservation
Virginia-Maryland Regional College of Veterinary Medicine
Biomedical Sciences and Pathobiology
Equine Medical Center
Large Animal Clinical Sciences
Small Animal Clinical Sciences
Maryland Campus
Veterinary Medicine Teaching Hospital
Apart from these schools and colleges, Virginia Tech has following 7 major research centers:
Institute for Society, Culture, and Environment (ISCE)
Fralin Life Science Institute
Institute for Creativity, Arts and Technology
Institute for Critical Technology and Applied Science (ICTAS)
Virginia Bioinformatics Institute at Virginia Tech
Virginia Tech Transportation Institute (VTTI)
Virginia Tech Carilion School of Medicine and Research Institute (VTCRI)
Ranked 69th among National Universities (the U.S. News & World Report)
Ranked 30th among National Public Universities (the U.S. News & World Report)
Positioned 7th among engineering schools at public universities


text:
Adaptations | Cartoon | Comedy | Family | Sci-Fi/Fantasy | TV spin-offs
Posted on October 27, 2019 December 25, 2019 by Joanna Berry
Is the animated version of The Addams Family as creepy, kooky and spooky as the 1991 live action classic?
Voices of: Oscar Isaac, Charlize Theron, Chloe Grace Moretz, Finn Wolfhard
Created by cartoonist Charles Addams in 1938, The Addams Family began life as a newspaper cartoon in the New Yorker before being adapted into a black and white television series in 1964.
The adventures of a creepy, spooky and kooky family with a love for the macabre was then rebooted in the early seventies as an animated TV show before being translated into a terrific live action movie in 1991 (you can read the Movies4Kids review here).
The family – glamorous mother Morticia, quirky father Gomez, kids Wednesday and Pugsley, Uncle Fester, butler Lurch and disembodied hand Thing – are so darkly fun it is no surprise that they have returned to the screen once more, this time in an animated adventure with a starry voice cast.
In this adaptation in which the characters look nicely similar to the original drawings, we see how the family got together and ended up living in isolation in an abandoned asylum in New Jersey. However, glimpses of the real world occasionally reveal themselves and Wednesday decides she wants to attend a regular school much to the horror of her parents, while local TV personality Margaux wants the family to leave as they threaten her perfect planned community of 'Assimilation'.
While there are some moments that will make young kids giggle, there's surprisingly few laughs for older kids and adults, and none of the deliciously dark creepiness and oddness we expect from the bizarre family. The success of movies like Hotel Transylvania, Despicable Me and ParaNorman prove that children love the darkly comic and twisted moments just as much as grown-ups, so it is a real shame – especially considering the wonderful source material of Charles Addams' cartoons – that there aren't any ghoulish delights here.
Is The Addams Family (2019) suitable for kids? Here are our parents' notes...
The film has some dark themes, but nothing that should bother the over-7s.
The story includes a guillotine over Wednesday's bed, crossbows, swords, explosives and references to death.
Young children may not like a scene at school in which Wednesday and her classmates are supposed to dissect frogs, only for Wednesday to bring them to life to attack a school bully.
Young children may find Thing, Lurch, Fester and Grandma a little scary.
The Addams Family – first trailer!
Monster Family review
If you like this, why not try:
Monster Family
Tags: Addams Family, animation, Charles Addams, Charlize Theron, Chloe Grace Moretz, comedy horror, Gomez, Lurch, Morticia, Oscar Isaac, Pugsley, Thing, Wednesday
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Matiullah Joyia wants to open a mosque, but for now he's looking for followers at swap meets, Starbucks and a women's jail.
"War," one of them said.
It's the type of reaction that compels Joyia, a missionary from the Ahmadiyya Muslim Community, to go to the jail every Sunday afternoon. He's looking to dispel myths about Islam, and to peacefully invite people into his religion.
"Anyone is welcome to stay, anyone is welcome to leave. It's a matter of the heart," Joyia said.
Matiullah Joyia begins a meeting at the Women's Community Correctional Center with a silent prayer.
It's also why he posts "Coffee, Cake and Islam" events on meetup.com, welcoming anyone who's interested to sit with him at a Starbucks and discuss Islam.
And it's why he sets up a booth filled with books on Islam at the Aloha Stadium Swap Meet.
When a family passing his booth at the swap meet approached him aggressively to insist, "Jesus is the only way," Joyia said he engaged them in conversation.
Still, these can be tough crowds for Joya, who's been working in Hawaii for a year and a half to generate a tiny congregation of three families. And his efforts don't have the support of the wider Islamic community.
About 4,500 Muslims live in Hawaii, according to Hakim Ouansafi, chairman of the Muslim Association of Hawaii. But in Ouansafi's opinion, Joyia isn't one of them.
"They're preaching not what Islam teaches but what (Mizra) Ghulam Ahmad, the founder of Ahmadi, teaches," Ouansafi said.
Mizra Ghulam Ahmad, who Ahmadis consider to be a messiah, founded the religion in 1889 in what is now Punjab, India. Their belief that Ahmad is a prophet separates Ahmadis from other branches of Islam.
The Ahmadiyya community is familiar with conflict. Its members have faced persecution and discrimination within the Islamic world for more than a century as a result of their doctrinal differences with mainstream Islamic sects.
In some Muslims-majority countries, Ahmadis are considered heretics and in some cases "worse than infidels," Joyia said. State-sponsored violence against the community, particularly in Indonesia, Bangladesh and Pakistan, has caused a mass exodus to Western countries.
In 1974, Pakistan's then-Prime Minister Zulfikar Bhutto amended the country's constitution to declare Ahmadis to be non-Muslims. Legislation passed a decade later made it criminal for Ahmadi people to proselytize or even identify as Muslim.
Joyia's own family fled Pakistan when he was 8 in search of religious freedom in Canada.
"We wanted to live in a peaceful place that offers us security," he said.
Joyia is one of about 2,000 official missionaries from the Ahmadiyya community who, following the completion of their religious education, are sent around the world.
"We've had some Ahmadis over the years in the Hawaiian Islands but we've never really had a formal chapter or presence," said Amjad Mahmood Khan, a national spokesperson for the Ahmadiyya Muslim Community in the United States.
Ahmadiyya is a centralized organization with 74 chapters in the United States and an international headquarters in London. Member contributions finance the sect's missionary work, including living expenses and a small stipend that Joyia and his family depend on for their livelihood.
Joyia arrived in Honolulu with his wife and daughter a year and a half ago after working for five years in the Marshall Islands, where he opened the country's first mosque and amassed a following of about 200 converts.
With just three Oahu families registered in the religious organization's national database, Joyia's local congregation is still in its infancy.
Imam Matiullah Joyia offers one-on-one lessons to a family at their home in Honolulu.
"It's hard to get people's attention because everybody's so busy making ends meet," he said.
After months of searching for a location, Joyia found a space to rent in Iwilei where he plans to open a mosque in the coming year. He hopes it will become a gathering place for Ahmadis and a hub for community service programs.
For now, he visits with the families individually to offer lessons and guide prayer.
At least initially, Joyia said he doesn't get into the theological differences between Ahmadis and other mainstream Muslims when he teaches. He said he preaches Islam, not Ahmadiyya theology, and lets his students choose which doctrine they agree with.
HaneefBilal Salahuddin Abdul-Shafiq, a social worker in the women's correctional center, said Joyia has had a positive impact on the inmates he's worked with.
Last year about 40 women fasted during Ramadan: "no food, no water, no chewing gum, no nothing," he said.
"In Hawaii … there's never been Islam in the women's prison, but we have it now," he said.
Audio reporting by Emily Dugdale.


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Editorials May 27, 2013 June 18, 2014
Julia on the F Train by Elizabeth Foster
Elizabeth Foster is not only a photographer, but also a poet, and this is apparent in his work. There is something so raw about each of the images that Foster captures. Atwood has worked with him many times and we always look forward to the final product. He takes risks, he's brave, and that's why we love him.
Tell us about yourself.
I'm a 21 year old photographer and poet currently living in Brooklyn, NY. I shoot almost exclusively on film. Although, I play around a lot with iPhone photography whenever I don't have my camera on me. I have seen a lot of people giving others slack for that, but I think it's great for capturing moments when I'm just walking around the city.
At what moment in your life did you decide to become a photographer?
I don't think I ever really had an epiphany or anything like that. I have been shooting film since I was in middle school. I really enjoyed how I could create images that I could share with other people taken from my own perspective. When I moved to New York City summer 2012 I become more focused on my goal of being published. For a while I was too afraid to submit to magazines because I was certain I would be rejected. I think moving here gave me the push I needed to realize that putting myself out there is always worth it even when I don't get the response I was hoping for.
What's the most powerful photograph you've seen? What makes it so powerful?
I recently saw a series shot by a trans* couple Rhys Ernst and Zackary Drucker documenting their transitions. It was beautiful. I actually cried when I saw the photographs. As someone who is struggling with my own issues with gender identity it really touched me on a personal level. It made me feel like even though what I am going through is painful at times I am by no means alone. It just felt so important.
What's something people would never expect about you?
I suppose that I'm trans. I identify as a boy. I present as very femme at times and a lot of people seem to have a hard time processing the concept that someone's outer appearance doesn't determine their gender identity.
View more of his work here.
Model: Julia Cumming//Photographer: Elizabeth Foster
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Jeremy Warren
Jeremy Warren originally from Jonesboro, Louisiana moved to Nashville in January of 05 to pursue his dream of music. He has since worked his way up to being a full time Professional drummer in the Nashville music scene. Jeremy has toured the United States with CCM artists and country artists. He has also logged many studio hours recording on projects ranging from country, gospel, blues, rock, funk, Christian rock, and everything in between. He continues to be a solid player working with some of the best musicians in the world.
Toured or Recorded with:
Sidewalk Prophets CCM (2010dove winning artist), Red Marlow, Monument QT, The Noise (2010 release), HWY 96 (soul/gospel 2010 release),Lee Roy Parnell, Wade Hayes, Craig White, Masters Voice, and tons of writers. I have also did many single Showcase dates for country artist and writers. I have also worked on many demo sessions for songwriters. Also worked on TV/Film sessions. Playing on tracks to be used in commercials and movies, TV movies. In late 2010 I was hired by Broken Bow country artist James Welsey!
Jeremy is also endorsed by SilverFox sticks, Attack heads.
Jeremy was introduced to Soultone Cymbals through Soultone endorser Mark Beckett, who is a top call session and Opry drummer.
"I continue to grow my love for music, mature as a musician, and do what I have always dream of doing since I was a kid in a small town in north Louisiana. I am truly blessed!!


Hannas Run Farm is a family farm. No chemicals or hormones added. Angus beef grain feed with our own farm grown corn. Whole, half, or quartered cut to your specifications, packaged and ready for the freezer.


IMPORTANT: This early demo version of FireSim is no longer supported. Instead, you should use the full open-source release of FireSim, which you can find more information about here: https://docs.fires.im.
We're excited to release our first public demo of FireSim, which you can deploy now on Amazon EC2 F1 instances by following the instructions on this page. Also, stay tuned for an upcoming AWS Compute Blog post about FireSim, which covers more details about FireSim and this demo. You can track FireSim development by following @firesimproject on Twitter.
By the end of this demo, you'll have an FPGA-accelerated cycle-accurate simulation of 1 or 8 RISC-V Rocket Chips, each with a NIC and block device, and interconnected by a functional network simulation. These simulated processors will boot a pre-built Linux distro included in the FireSim Demo AMI. At the end, we'll run memcached on the simulated nodes and run YCSB on the EC2 instance to generate load on our simulated cluster of Rocket Chips and demonstrate the ability to run real workloads on FireSim. Running this demo does not require any knowledge about FPGAs or RISC-V.
There are two ways to try the demo, either simulating a single node or simulating an 8-node cluster, which require either an f1.2xlarge or an f1.16xlarge instance respectively. The instructions will guide you to the appropriate section based on your choice.
We provide a pre-built solution on the AWS Marketplace that includes an AMI/AFI combo to deploy the simulation. You can find it here. To simulate a single node, start an f1.2xlarge instance using our AMI. To simulate an 8-node cluster, start an f1.16xlarge instance using our AMI. Below are detailed instructions for setting up an instance for new users. If you've used F1 instances before, you can skip to the next section after starting an instance.
If you are a new EC2 F1 user, open the AWS EC2 Management Console. In the top right corner, make sure that your region is set to N. Virginia (a.k.a. us-east-1). This is required to be able to use F1 instances. If you are a new EC2 user, it is also likely that your service limit for F1 instances will be set to zero. You can check your limit at this link. Confirm that the limit for f1.2xlarge instances is greater than zero. If it isn't, follow the instructions to submit a service limit increase here to request access to F1 instances. Set an initial request size of 1 for either f1.2xlarge or f1.16xlarge instances in N. Virginia. You can put "FireSim HW Simulation" for the use case. This request will need to be approved before you can proceed.
Next, you can launch an instance using the FireSim Demo AMI. To launch, click "Continue" then "Launch with 1-click" on that page. The default options are sufficient to get started, but you may configure them as you wish. One option you may wish to change is to use an f1.16xlarge instance if you want to run an 8-node cluster of Rocket Chips.
Once your instance has booted, login with username centos and the key you supplied at instance creation time, just like the FPGA Dev AMI. The first time you login, you should see the regular FPGA Dev AMI login message, in addition to a message that says "FireSim network config completed." This sets up the necessary tap interfaces and bridge to enable communicating with the simulated nodes from the EC2 Instance.
riscv64-unknown-* toolchain: The riscv-tools toolchain is pre-installed, including gcc and binutils. You can see all of the included tools by typing riscv64-unknown- and hitting tab twice.
~/firesim-target-software: A custom-built Linux Distribution that runs on the simulated nodes. This directory contains a file called bbl-vmlinux, which contains the bbl bootloader and a Linux kernel. This directory also contains 8 root filesystem images, rootfs[0-7].ext4, one for each simulated node. If these images become damaged or you want to add additional software to the image, see the Extras section for how to re-build them.
FireSim-f1: This program controls simulation and communicates with the FPGA. We will not invoke this directly in this demo, instead opting to use convenience scripts that automatically pass command line arguments to this program.
boot-firesim-singlenode and boot-firesim-cluster: These scripts automatically invoke FireSim-f1 with the appropriate arguments to boot Linux with the appropriate root filesystem images. The singlenode script runs a single simulation, while the cluster script runs an 8-node simulation. The cluster script can be run only on an f1.16xlarge instance.
To familiarize ourselves with the environment, we will first simulate a single node. The single node simulation will work on both f1.2xlarge instances and f1.16xlarge instances.
At this point, we know the simulated node is online. We can ssh into it using the username root and password firesim. It is also convenient to make sure that your TERM variable is set correctly. In this case, the simulation expects TERM=linux, so we will provide that.
Warning: Permanently added '192.168.1.10' (ECDSA) to the list of known hosts.
At this point, you can run programs on the simulated node, as you would with a real machine. For example, you can jump down to section 5 below to run YCSB against memcached on the simulated node.
You can confirm that the simulation has ended by running screen -ls, which should now contain no detached screens.
If you are running on an f1.16xlarge instance, we can continue on to simulate a cluster of 8 RocketChips. If you followed the previous steps to run a single node, make sure you have powered off the old simulated node first. If you are running an f1.2xlarge instance, skip this step and jump to step 5.
To start the 8-node cluster simulation, run the following.
Once all the nodes have booted, you should be able to ping any node from any other node. As before, you can shut down individual simulated nodes with poweroff. You can check which nodes are still running by checking for entries in screen -ls.
As a sample workload, we'll now run the Yahoo! Cloud Services Benchmark (YCSB) against our cluster of one or 8 RocketChips. Be sure to start memcached as shown above on each simulated node.
To load the data needed for YCSB, run the following commmand. If you're running a single simulated node, remove the IP addresses for nodes 1 to 7 (IPs 192.168.1.11 to 192.168.1.17) from the commands.
This will produce output showing you the real-time performance of the simulated cluster in serving memcached requests to the outside world. For a more visual output, you can install iftop and run sudo iftop -n -i br0 to see a visualization of traffic between the EC2 instance and the simulated nodes.
This will rebuild all of the rootfs[0-7].ext4 to include your new packages.
To resolve this, simply ping the node from the EC2 instance as the guide indicates. Linux boot will then continue.
We'll add more troubleshooting steps here as we receive feedback about particular issues.
Post on the FireSim Google Group and we'll help you fix your problem.
FireSim 1.4.0 released with new debugging tools!
Just in time for our tutorial at the first Chisel Community Conference, we've released a new version of FireSim that adds lots of new features! The headlining features with this release are new debugging tools, including assertion synthesis and Rocket Chip/BOOM commit log tracing. We've also bumped to more recent versions of Rocket Chip/BOOM, updated the block device model to be deterministic, started preparing infrastructure necessary to merge FireSim's "supernode" mode to master, and done lots of other internal cleanup. See the full changelog on GitHub.


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Cybersecurity (0) Apply Cybersecurity filter
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Born Digital: Privacy Introduction to Privacy Berkman Klein Center's Youth and Media Academia English 2010 Privacy and Reputation Identity Exploration and Formation, Information Quality https://www.youtube.com/watch?v=P0E4mVdMpcM Video
Cambodia: 'Avatar' Rally to Protect Country's Forest Pop Imagination Sopheap Chak (Global Voices) Non-profit
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Password Strength Passwords xkcd Independent English 2011 Security Content Production, Information Quality, Privacy and Reputation https://xkcd.com/936/ Visual
Revealing the Man Behind @MayorEmanuel Who Do You Want To Be? Alexis Madrigal (The Atlantic) Private English 2011 Identity Exploration and Formation Content Production, Context, Digital Economy, Privacy and Reputation https://www.theatlantic.com/technology/archive/2011/02/revealing-the-man-behind-mayoremanuel/71802/ Text


Last major update issued on December 11, 2003 at 04:40 UTC.
The geomagnetic field was active to major storm on December 10. Solar wind speed ranged between 586 and 879 km/sec under the influence of a high speed stream from coronal hole CH71.
Solar flux measured at 20h UTC on 2.8 GHz was 89.2 (this is the lowest solar flux at 20h UTC since March 22, 2003. Adjusted to 1 AU solar flux was 86.5, the lowest since February 2, 1998). The planetary A index was 42 (STAR Ap - based on the mean of three hour interval ap indices: 43.0).
Three hour interval K indices: 54565554 (planetary), 63554544 (Boulder).
Region 10517 decayed further and will rotate over the southwest limb on Dec.12.
New region 10520 rotated into view at the northeast limb early in the day.
New region 10521 rotated into view at the southeast limb early in the day.
December 8-10: No partly or fully earth directed CMEs observed.
A huge recurrent trans equatorial coronal hole (CH71) will be in a geoeffective position on December 5-11. Some decay was observed in the northern and western parts on December 9-10.
Processed SOHO/EIT 284 image at 19:06 UTC on December 10. The darkest areas on the solar disk are likely coronal holes.
The geomagnetic field is expected to be unsettled to minor storm with occasional major storm intervals until December 14 or 15 due to a strong high speed stream from coronal hole CH71.
Long distance low frequency (below 2 MHz) propagation along east-west paths over high and upper middle latitudes is poor to fair. Propagation along long distance north-south paths is fair to good. [Trans Atlantic propagation conditions are currently monitored every night on 1470 kHz. Dominant stations tonight: None. A number of stations were heard at various times: Radio Cristal del Uruguay, Radio Rafaela (Argentina), another unidentified station from Argentina and these stations from Brazil: Rádio Papacaça AM, Rádio Jornal Fluminense and Rádio Morro Verde. On my antenna to the west WLAM Lewiston ME with their WMTW relay was noted at times. Several other east coast US stations could be heard despite the auroral conditions, e.g. WTOP Washington DC on 1500 and WWZN Boston MA on 1510 kHz].


On the surface, it doesn't look like the Tigers got much in their deal for Nate Robertson. They had to pay almost all his salary and got a mid grade prospect projected as a lefty specialist out of the bullpen in return. Jay Voss an 8th round pick in 2007 had decent stats in AA last year but seems to be far below the original expectation the Tigers had in a deal for Robertson. Since they had to pay most of his salary anyway, I guess they didn't feel he would perform enough to be at least a B level free agent in which they'd pick up a draft pick better than Voss.
With so many teams at or close to the max of 40, including the Jays, teams have little room to add a player, without risking losing someone else. The roster sizes according to mlb.com at the moment. Note that players on the 60 day DL do not impact the 40 man roster.
For the Jays this should not be a problem since anyone they trade will likely come from the 40 man pitching roster anyway and for which we have a suitable replacement. My expectation is that whoever the Jays acquire will be a prospect that may not yet be required to be put on the 40 man roster. So we could effectively free up a spot or two on the roster through trade.


prescription & nonprescription eyewear, accessories, repairs and adjustments.
Eyes On Main Ltd was founded in 2006. The company is located in North Andover and incorporated in Massachusetts. Eyes On Main Ltd specializes in Optical Goods Stores.
Eyes On Main LTD is located at 75 Main St, North Andover, MA. This business specializes in Opticians.


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/**
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package org.apache.reef.wake.metrics;


Presenters for each of the leadership skills workshops will be providing us with links to helpful resources on their topic. Resources will be added as we receive them, on an ongoing basis. Please check back regularly.
Chapters 3 and 5 from Delee Fromm, Advance Your Legal Career: Essential Skills for Success.


This is just one of several football apps - part of the Football PRO membership.
Whether you are a receiver, running back, or lineman - speed matters the most. This program includes the most advanced football speed and agility exercises used by pro and collegiate football players.
Develop the muscles to become more shifty, accelerate in the open field, and improve leg drive.
The top pro football players use these drills to prepare for the combine in order to get an advantage and propel their game to the next level. Football becomes much easier when you can run faster and are quicker than your opponent.
Become a PRO or MVP member to get access to more Fitivity apps, plus other features and advantages!
Access all Fitivity Football related apps and their premium levels.
Access to the entire collection of 350 Fitivity apps and their premium levels.
- Your own dedicated training library to access thousands of videos, exercises, and workouts.
- The ability to create and edit your own training library.
- Save exercises and workouts for easy access.
- A timer so you never have to leave the app to time your exercises.