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Enterotoxigenic Escherichia coli (ETEC) are common causes of diarrheal morbidity and mortality in developing countries for which there is currently no vaccine. Heterogeneity in classical ETEC antigens known as colonization factors (CFs) and poor efficacy of toxoid-based approaches to date have impeded development of a broadly protective ETEC vaccine, prompting searches for novel molecular targets.
Using a variety of molecular methods, we examined a large collection of ETEC isolates for production of two secreted plasmid-encoded pathotype-specific antigens, the EtpA extracellular adhesin, and EatA, a mucin-degrading serine protease; and two chromosomally-encoded molecules, the YghJ metalloprotease and the EaeH adhesin, that are not specific to the ETEC pathovar, but which have been implicated in ETEC pathogenesis. ELISA assays were also performed on control and convalescent sera to characterize the immune response to these antigens. Finally, mice were immunized with recombinant EtpA (rEtpA), and a protease deficient version of the secreted EatA passenger domain (rEatApH134R) to examine the feasibility of combining these molecules in a subunit vaccine approach.
EtpA and EatA were secreted by more than half of all ETEC, distributed over diverse phylogenetic lineages belonging to multiple CF groups, and exhibited surprisingly little sequence variation. Both chromosomally-encoded molecules were also identified in a wide variety of ETEC strains and YghJ was secreted by 89% of isolates. Antibodies against both the ETEC pathovar-specific and conserved E. coli antigens were present in significantly higher titers in convalescent samples from subjects with ETEC infection than controls suggesting that each of these antigens is produced and recognized during infection. Finally, co-immunization of mice with rEtpA and rEatApH134R offered significant protection against ETEC infection.
Infectious diarrhea is one of the leading causes of death among young children in developing countries, and a major cause of morbidity in all age groups. The enterotoxigenic Escherichia coli contribute substantially to this burden of diarrheal illness, and have been a focus of vaccine development efforts for more than forty years following their discovery as a cause of severe diarrheal illness. The heat-labile, and/or heat stable enterotoxins that define ETEC are produced by a diverse population of Escherichia coli. This inherent genetic plasticity of E. coli has made it difficult to identify antigens specific to ETEC that are highly conserved. Therefore, identification of protective antigens shared by many ETEC strains will likely play an essential role in development of the next iteration of vaccines.
Citation: Luo Q, Qadri F, Kansal R, Rasko DA, Sheikh A, Fleckenstein JM (2015) Conservation and Immunogenicity of Novel Antigens in Diverse Isolates of Enterotoxigenic Escherichia coli. PLoS Negl Trop Dis 9(1): e0003446. https://doi.org/10.1371/journal.pntd.0003446
Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, UNITED STATES
Received: August 8, 2014; Accepted: November 26, 2014; Published: January 28, 2015
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication
Data Availability: Data for this manuscript can be found in the manuscript itself, in Supporting information or in online public repository at NCBI. Individual URLs for data are provided in the body of the manuscript.
Funding: These studies were funded in part by grant R01AI89894, and contract HHSN272200900009C from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (http://www.niaid.nih.gov); the Department of Veterans Affairs, Office of Research and Development (http://www.research.va.gov); and the Bill & Melinda Gates Foundation (OPP1099494). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of any of the sponsoring agencies.
Competing interests: The authors have declared that no competing interests exist.
The enterotoxigenic Escherichia coli (ETEC) are among the most common causes of infectious diarrhea worldwide. Importantly, ETEC are disproportionately represented in cases of severe diarrheal illness as well as in deaths due to diarrhea among young children in developing countries .
These pathogens cause diarrhea by the elaboration and effective delivery of heat-labile and/or heat-stable enterotoxins to intestinal epithelial cells where they stimulate production of cyclic nucleotides ultimately activating the cystic fibrosis transmembrane regulator (CFTR) with resulting net efflux of fluid into the intestinal lumen. Plasmid-encoded colonization factors (CFs), discovered  shortly after these organisms were identified as a causative agent of cholera-like diarrheal illness[4–6], are thought to be essential for effective colonization of the small intestine and required for ETEC pathogenesis.
Following early studies suggesting a pivotal role for these structures[7,8], CF antigens have defined the basis for most subsequent ETEC vaccine efforts [9,10]. However, one factor complicating development of a broadly protective vaccine for ETEC has been the general plasticity of E. coli genomes, and the significant antigenic heterogeneity of the CFs. To date, at least 26 antigenically distinct CF antigens have been described. The lack of appreciable cross-protection afforded by these antigens combined with the complex landscape of CFs portrayed in ETEC molecular epidemiology studies continue to complicate rational CF antigen selection.
Antigenic heterogeneity, recent failure of LT-toxoid-based vaccine strategies[14,15], as well as the need to optimize the performance of live-attenuated vaccines currently in clinical trials [16–18] have highlighted the need to identify additional virulence molecules that might be targeted in ETEC vaccines. Recent efforts led to the identification of two loci discovered on the same virulence plasmid of ETEC strain H10407 that encodes the CFA/I colonization factor. These include the etpBAC two partner secretion locus responsible for production and export of EtpA, a novel adhesin molecule which bridges highly conserved regions of flagellin and the eukaryotic cell surface. Also located on this plasmid is the eatA gene that encodes the EatA serine protease autotransporter molecule capable of degrading EtpA as well as MUC2, the major gel-forming soluble mucin in the small intestine.
Recent immunoproteomic and transcriptomic  analyses of H10407 have also highlighted two chromosomally encoded antigens that are not specific to the ETEC pathovar, but which nonetheless appear to be involved in the pathogenesis of these organisms. Conceivably, these molecules, YghJ, a secreted mucin-degrading metalloprotease, and EaeH , an adhesin, act in concert with colonization factors and other pathovar-specific virulence proteins like EatA and EtpA to promote toxin delivery.
While emerging data suggests that these novel proteins are highly immunogenic and that EtpA and EatA are protective antigens[29–31] in a murine model of ETEC infection, additional data regarding their conservation among ETEC strains are needed to determine their suitability as vaccine targets. Here we demonstrate that these antigens are broadly represented in a diverse collection of ETEC isolates suggesting that they could be employed to augment existing approaches to ETEC vaccine development.
Materials and Methods
Bacterial strains and growth conditions
ETEC strains used in this study are detailed in S1 Dataset. All strains were grown at 37° in Cassamino acids yeast extract media (CAYE: 2.0% Casamino Acids, 0.15% yeast extract, 0.25% NaCl, 0.871% K2HPO4, 0.25% glucose, and 0.1% (v/v) trace salts solution consisting of 5% MgSO4, 0.5% MnCl2, 0.5% FeCl3) from frozen glycerol stocks maintained at −80°C.
Strain characterization by disease severity and colonization factor type
Strains from the International Centre for Diarrhoeal Disease Research (icddr,b) in Dhaka were selected based on their associated disease severity using modified WHO guidelines as previously outlined. Expression of individual CFs was determined by dot immunoblotting with monoclonal antibodies specific to each respective CFs (CF-MAb) as previously described . Briefly, 2 μl of a PBS suspension containing ∼106 colony forming units of each ETEC strain was dotted onto nitrocellulose, air-dried, blocked with BSA in PBS, followed by detection with CF-MAbs and goat anti-mouse IgG_HRP conjugate. Bound MAbs were then detected with 4-chloro-1-naphthol chromogen and H2O2.
Screening for ETEC virulence genes by PCR
We screened a total of 181 ETEC available isolates currently maintained as frozen glycerol stocks in our laboratories. The majority of these strains were collected between 1998 and 2011 in Bangladesh, and were obtained from the icddr,b in Dhaka. Complementing this collection were geographically disparate strains associated with severe diarrheal illness including strains from the Amazon region in Brazil , and ThroopD, an isolate from a patient with severe ETEC diarrheal illness who presented in Dallas in the 1970s. Strains encoding eatA and etpA were identified by PCR using primers directed against conserved regions of these genes as previous described . Briefly, a small amount of frozen glycerol stock from each strain was introduced with a sterile pipette tip into a PCR mixture containing the respective primers and a master mix. Toxin genotypes were confirmed in these isolates using multiplex PCR screening for genes encoding heat-labile (LT), and heat-stable toxins (STp, and STh) as previously described. Primer sequences are listed in S1 Table.
Immunoblotting for secreted ETEC virulence antigens
To determine production of secreted virulence antigens by different ETEC strains, supernatants from overnight cultures were first precipitated with trichloroacetic acid (TCA)  and resuspended in sample buffer before polyacrylamide gel electrophoresis. Western blotting was then performed using polyclonal rabbit antisera against recombinant versions of either EatA, EtpA, or YghJ that were pre-absorbed against an E. coli lysate column (Pierce) and affinity-purified using the antigen immobilized on nitrocellulose membranes as previously described [31,38], followed by detection with affinity-purified secondary goat anti-rabbit-(IgG)-HRP conjugate (Santa Cruz Biotechnology, SC2004).
Protein sequence comparisons of ETEC pathovar specific antigens
To examine antigenic conservation of EatA among ETEC isolates for which genomic DNA sequences are currently available, BLASTP was used to search GenBank https://www.ncbi.nlm.nih.gov/genbank/ using the full length sequence of the EatA protein from strain H10407 (https://www.ncbi.nlm.nih.gov/protein/AAO17297.1) as the query sequence. To construct alignments of EatA from positive strains, the 1042 residue passenger domain (corresponding to amino acids 57–1098 of EatA from H10407) was compared with EatA of ETEC isolates derived from different phylogenic lineages using a CLUSTAL Omega (release 1.2.0 AndreaGiacomo)  algorithm plugin for CLC Main Workbench v6.9.1. A similar approach was used to compare the amino-terminal sequence of EtpA (amino acids 1–600, GenBank accession number AAX13509.2).
Conservation heat mapping
Virulence protein expression data from the collection of 181 strains under study were included in the analysis. Heat maps were configured using R version 3.1.0 (2014, http://www.R-project.org/) using gplots and RColorBrewer packages installed from http://CRAN.R-project.org using the heatmap2 function within gplots (see S2 Dataset).
Recombinant protein production
The antigens used in these studies were produced as polyhistidine-tagged recombinant proteins and purified by immobilized metal ion affinity chromatography (IMAC) as previously described[27,29,44,45]. Additional polishing steps including size exclusion or ion exchange chromatography were performed as needed to produce highly purified antigens. Purity of each antigen was assessed by SDS-PAGE followed by sensitive Coomassie Blue staining. Purified recombinant antigens were stored at −80°C.
Assessment of immune responses to novel ETEC virulence proteins
To quantitfy antibody concentrations directed at novel recombinant antigens, kinetic ELISA was performed on dilutions of plasma samples previously obtained from patients hospitalized at the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (icddr,b) with acute symptomatic ETEC infections. Plasma samples from non-infected adults and children obtained at icddr,b, or specimens obtained from children at Saint Louis Children’s Hospital were used as negative controls. Samples from human volunteer ETEC H10407 challenge studies were kindly provided by Dr. Robert Gormely and Dr. Stephen Savarino of National Naval Medical Center, Bethesda Maryland.
Use of these clinical materials was approved by the Institutional Review Boards of both icddr,b and Washington University School of Medicine. All plasma samples were maintained at 4°C in a humidified chamber prior to use in ELISA. Immune responses to purified recombinant proteins (rYghJ, rEaeH, rEtpA, rEatAp) were assessed by kinetic ELISA as previously described [30,47]. Antigen binding to ELISA wells (Corning, Costar 2580) was first optimized to determine the optimal coating concentration and buffer system, using highly antigen-specific polyclonal rabbit antisera to detect binding by ELISA. Purified antigens were then diluted either in 50 mM carbonate buffer (pH 9.6) (rEtpA-myc-His6, 1 μg/ml; rEatAp, 10 μg/ml; rYghJ-myc-His6, 1 μg/ml); or in phosphate buffered saline (PBS, pH 7.4) (rEaeH-myc-His6, 1 μg/ml). ELISA plate wells were coated with 100 μl/well overnight at 4°C, washed with PBS containing 0.05% Tween-20 (PBS-T), and blocked for 1 h at 37°C with 1% BSA in PBS-T. All plasma samples were diluted at 1:4096 in blocking buffer. After incubation for 1 hour at 37°C, plates were washed with PBS-T, and secondary goat anti-human IgG(H+L)-HRP conjugated antibody (Pierce, 31410) was added at a final concentration of 1:10,000. After incubation for 30 minutes at 37°C, plates were washed and developed with TMB microwell peroxidase substrate [3,3’,5,5’-Tetramethylbenzidine] (KPL, 50-76-00). Kinetic absorbance measurements were determined at a wavelength of 650 nm, and acquired at 40 s intervals for 20 minutes using a microplate spectrophotometer (Eon, BioTek). All data were recorded and analyzed using Gen5 software (BioTek) and reported as the Vmax expressed as milliunits/min. Statistical calculations were performed using Prism v4.0c (GraphPad Software), using nonparametric Mann-Whitney (two-tailed) comparisons of data.
Mouse immunization and challenge studies
These studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, using an established protocol approved by the Washington University School of Medicine Animal Studies Committee.
Four groups of twelve CD-1 mice were immunized intranasally with either 1 μg of LT (adjuvant only controls), or 1 μg of LT + 15 μg of rEatAp(H134R), or 1 μg of LT + 15 μg of rEtpA, or 1 μg of LT + 15 μg of rEatA(H134R)+15 μg of rEtpA on days 0, 14, 28. On day 40, mice were treated with streptomycin [5 g per liter] in drinking water for 24 hours, followed by drinking water alone for 18 hours. After administration of famotidine to reduce gastric acidity, mice were challenged with 106 cfu of the kanamycin-resistant (lacZYA::KmR) strain jf876 by oral gavage as previously described. Fecal samples (6 pellets/mouse) were collected on day 42 before oral gavage, re-suspended in buffer (10mM Tris, 100mM NaCl, 0.05% Tween 20, 5mM Sodium Azide, pH 7.4) overnight at 4°C, centrifuged to pellet insoluble material, and recover supernatant for fecal antibody testing (below). Twenty-four hours after infection, mice were sacrificed, sera were collected, and dilutions of saponin small-intestinal lysates were plated onto Luria agar plates containing kanamycin (50 μg/ml).
Murine immune responses to LT, EatA and EtpA were determined using previously described kinetic ELISA. Briefly, ELISA wells were coated with 1 μg/ml GM1, or 10 μg/ml of rEatAp(H134R), or 1 μg/ml rEtpA in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3, pH8.6) overnight at 4°C. Wells were washed three times with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T), blocked with 1% bovine serum albumin (BSA) in PBS-T for 1 h at 37°C, and 100 μl of fecal suspensions (undiluted) or sera (diluted 1:100 in PBS-T with 1% BSA) was added per ELISA well and incubated at 37°C for 1 h. Horseradish peroxidase-conjugated secondary antibodies were used and signal detected with TMB (3,3′,5,5′-tetramethylbenzidine)-peroxidase substrate (KPL) substrate.
All animal studies were performed under protocols approved by the Animal Studies Committee of Washington University School of Medicine (protocol number 20110246A1). All procedures complied with Public Health Service guidelines, and The Guide for the Care and Use of Laboratory Animals.
All human studies included were performed under a protocol approved by the Institutional Review Board of Washington University School of Medicine (IRB ID# 201110126). All of the human studies here report anonymous analysis of de-identified pre-existing sera previously stored from earlier studies for which no additional consent was obtained.
Conservation of ETEC pathogen-specific secreted antigens
Two novel antigens, the EtpA adhesin, and the passenger domain of the EatA serine protease are encoded on the large 92 kilobase virulence plasmid of the prototypical ETEC strain H10407. Both of these secreted proteins[22,30] are required for H10407 to efficiently deliver heat-labile toxin to target epithelial cells. Furthermore, both of these antigens are immunogenic , and induce protective immune responses in a murine model of ETEC intestinal colonization[29,31]. To further assess their utility as potential vaccine antigens, we examined a large collection of ETEC strains that were well characterized with respect to associated clinical metadata pertaining to disease severity and which had not undergone repeated serial passage in the laboratory.
Altogether, we found that these antigens are relatively conserved in the ETEC pathovar, confirming the results of earlier studies that focused on strains from different phylogenies obtained in Guinea Bissau and Chile [37,49]. Of the 181 strains examined in the present study (Fig. 1), we found that more than half of all strains produced EtpA (102/181, 56%) and/or EatA (106/181, 59%) (S1 Dataset), and that more than three quarters of all strains produced at least one of these antigens. Both EtpA and EatA were identified more than twice as frequently as the most commonly identified CF (CS6), which was identified in 22% of strains in this collection (Table 1).
All of the strains in the collection (n = 181) were examined for production of three secreted ETEC virulence proteins EtpA, EatA, and YghJ by immunoblotting of culture supernatants with the respective antibodies. A subset of these strains (n = 91) were recently sequenced at the Genome Sequencing Center for Infectious Diseases (GSCID).
Relationship of plasmid-encoded virulence loci to colonization factor antigens
Importantly, although the genes encoding the etpBAC secretion system and the EatA autotransporter were initially discovered on the same large virulence plasmid of H10407, which also encodes the colonization factor (CF) CFA/I, we found that these loci were not restricted to strains expressing this particular CF, but were widely distributed among the different CFs, and were also present in strains for which no CF could be identified (Fig. 2A). Indeed, half of the strains for which no CF could be identified expressed either EtpA or EatA, suggesting that these antigens could complement existing vaccination strategies centered on CFs. As expected by the association with multiple CFs, we also found that EtpA and EatA were secreted by strains from multiple phylogenic lineages (Fig. 2B, C). Interestingly, however we found a negative association between the etpBAC locus and strains expressing CFA/IV antigens [50,51] including CS5 in that none of the 23 strains possessing CS5 fimbriae secreted the EtpA adhesin. Similarly, among strains expressing CS6, which is frequently co-expressed with CS5, only a minority secreted EtpA. These data are also consistent with our earlier observation that the prototype B7A strain, which also expresses CS6, lacks the etpBAC locus and does not secrete EtpA.
a. Heatmap of EtpA and EatA showing the proportion of strains positive for expression of these antigens among different CF groups. CF antigen designation is shown at left of the heatmap. nd = no CF antigen detected. Below is the heatmap key depicting colors associated with each degree of antigen positivity. Density line in yellow depicts the relative number of map features assigned at each proportion. Primary data used to construct the heatmap can be found in S2 Dataset. b. Imunoblot detection of EtpA and EatA expression among strains from different phylogenies. The upper immunoblot demonstrates EtpA production in the prototype H10407 strain, ThroopD isolated in Dallas, TX in 1975, the Juruá_18/11 (Amazon, 1998), and phylogentically dispersed strains from icddr,b. The etpA mutant is included as a negative control. The lower blot demonstrates EatA production by H10407, phylogenically distributed strains from icddr,b and Envira_10/1, an additional isolate from cholera-like outbreaks in the Amazon. The eatA mutant is included as a negative control. c. Phlyogram showing the phylogenetic distribution of selected ETEC strains (designations in blue) and reference E. coli strains (designations in black). Red circles and gold stars represent eatA+, and etpA+ strains, respectively.
Interestingly, both the eatA and etpBAC loci were originally identified in ETEC strain H10407, originally isolated from an adult with severe, cholera-like illness in Bangladesh. As has been noted previously, this strain also causes more severe illness in human clinical challenge studies relative to other strains like B7A that lack these loci. Because we had clinical metadata pertaining to disease severity for all of the strains in our collection, we questioned whether the production of either of these antigens was associated with strains isolated from more severe forms of infection. However, we did not find any clear association between either of these putative virulence loci and clinical outcome (S1 Dataset).
Conservation of chromosomally-encoded antigens
We also examined the conservation of two chromosomally-encoded antigens which are not specific to the ETEC pathovar, but have recently been shown to play a role in virulence. The eaeH gene was originally identified on the chromosome of ETEC strain H10407 by subtractive hybridization with E. coli MG1655, is transcriptionally activated by cell contact , and under these conditions EaeH is produced by a diverse group of strains belonging to different phylogenies. Using the EaeH peptide sequence from H10407 (GenBank accession AAZ57201), BLASTP searches of recently sequenced ETEC strains from Bangladesh and elsewhere (http://gscid.igs.umaryland.edu/wp.php?wp=comparative_genome_analysis_of_enterotoxigenic_e._coli_isolates_from_infections_of_different_clinical_severity) also revealed that the eaeH gene was present in 63 out of 91 distinct isolates (69%) (S1 Dataset). BLASTP searches of these data for another chromosomally encoded molecule, YghJ, a type II secretion system effector recently shown to be involved in mucin degradation and toxin delivery demonstrated that the yghJ gene was present on the chromosomes in 83 of 91 (91%) isolates. Similarly, we identified the YghJ protein in a majority (161/181, 89%) of ETEC culture supernatants (S1 Dataset). This antigen was produced across ETEC strains expressing multiple CF types including 31/36 strains that were CF-negative by monoclonal antibody screening.
EatA and EtpA sequence conservation
Ideally, putative vaccine targets should be specific to the pathovar under study or restricted to pathogenic isolates, but not subject to significant antigenic variation. Therefore to further examine the potential utility of two ETEC pathovar specific antigens, EtpA and EatA, as vaccine candidates, we used recently obtained DNA sequence information from multiple ETEC genomes belonging to different phylogenies and from temporally and geographically disparate sources to compare the predicted amino acid sequences of these proteins.
For the prototype EatA molecule, first described in ETEC H10407, the 1042 residue region from amino acids 57–1098 is predicted for the secreted passenger domain that contains the serine protease catalytic triad as well as protective epitopes. We therefore compared this region of the molecule to those derived from the recently released genome sequences of multiple ETEC strains. Altogether, we found that the sequence of the EatA passenger domain (EatAp) was very highly conserved across strains, and exhibited between 95–100% identity to the prototype H10407 Eatp (Table 2). Likewise, the predicted serine protease catalytic motif formed by the histidine, aspartic acid and serine residues at positions 134, 162, and 267, respectively were universally conserved within the passenger domains of these proteins (S1 Fig.). Similarly, the predicted amino acid sequences of the secreted EtpA adhesin molecules from multiple strains exhibited between 94 and 100% identity to the H10407 prototype antigen (Table 3, S2 Fig.).
Despite the fact that the comparator strains included here spanned isolates collected over nearly 40 years, belonging to different phylogenies and that strains originated in diverse locations in Asia, Africa and the Americas, both proteins appear to exhibit remarkably little antigenic variation. Likewise, in analysis of the genomes of strains isolated recently within Bangladesh both proteins demonstrated similar degrees of sequence conservation (S1–S2 Fig.).
Immunogenicity of novel virulence antigens
Earlier immunoproteomic studies suggested that a variety of conserved E. coli proteins as well as ETEC pathovar specific proteins are recognized during the course of experimental infections in mice, and these responses parallel those observed using pooled convalescent sera from ETEC patients. To further characterize the immune response to novel antigens we focused on four proteins that have recently been shown to play a role in ETEC pathogenesis, including two plasmid-encoded secreted ETEC pathovar-specific antigens: EatA protease, and the EtpA adhesin, as well as the highly conserved chromosomally-encoded YghJ metalloprotease and the EaeH adhesin protein.
In comparing convalescent plasma from patients hospitalized at icddr,b to uninfected controls from Bangladesh, we found that patients in general exhibited significantly greater total antibody (IgG, IgM, IgA) responses to each of these antigens following diarrheal illness (Fig. 3) suggesting that these proteins are expressed during the course of infection. Similar results were obtained in comparing plasma from un-infected children from a non-endemic area in the United States (S3 Fig.).
We also examined the immune response to EtpA following infection by examining sera obtained before and after challenge of human volunteers with ETEC H10407. In sera obtained from two independent volunteer challenge studies, we also observed significant increases in immune responses to EtpA (S4 Fig.), strongly suggesting that this secreted protein is specifically recognized following infection by ETEC strains including H10407 that secrete this antigen.
Shown are kinetic ELISA data for four different recombinant antigens (a, rEtpA; b, the rEatA passenger domain; c, rYghJ; and d, rEaeH) obtained with 1:4096 dilutions of convalescent plasma from ETEC-infected patients hospitalized at ICDDR,B in Dhaka, Bangladesh (closed circles), or control patients not infected with enterotoxigenic E. coli (open circles). Horizontal bars represent geometric mean Vmax kinetic ELISA values for each group. P values obtained by two-tailed Mann Whitney testing of groups are summarized (*<0.05; **<0.01; ***<0.001;****<0.0001). x-axis of each graph depicts the specificity of the secondary antibody used in the ELISA (IgG, IgA, IgM, and total IgG, IgA, and IgM).
Protective efficacy of combined EtpA-mutant EatA passenger vaccination
Previous studies have demonstrated that individually, vaccination with either EtpA[31,56] or the passenger domain of the EatA serine protease affords protection against intestinal colonization in mice. The data above suggest that collectively these antigens might significantly extend coverage presently offered by classical approaches to ETEC vaccine development. We therefore questioned whether these two antigens could be successfully combined in a subunit approach. Because we have previously demonstrated that the native secreted EatA passenger domain will degrade intestinal mucin as well as the EtpA adhesin molecule, we elected to vaccinate animals with a modified recombinant version of the EatA passenger that lacks protease activity (rEatApH134R).
Co-vaccination with rEtpA and the mutant rEatApH134R molecule elicited robust serologic responses to both molecules that were comparable to vaccination with either antigen alone. As anticipated, each of the groups mounted strong serologic responses to the LT adjuvant (Fig. 4A), and both antigens retained their immunogenicity following co-immunization of EtpA with the rEatAH134R passenger domain (Fig. 4B,C) with responses that were at least comparable to those obtained following immunization with either antigen alone (Fig. 4C). Likewise, mice immunized with both antigens were significantly protected against colonization by ETEC (Fig. 4D), although co-vaccination with both antigens did not appear to be more effective than vaccination with either antigen alone. Collectively, however, these data suggest that co-immunization with these two antigens is feasible, and could be employed to expand present approaches to ETEC vaccine antigen selection.
Serologic responses to a heat-labile toxin (LT), b the passenger domain of EatA (Eatp), c EtpA. Shown are serum IgG responses following intranasal vaccination of mice with the LT adjuvant alone, or LT with 15 μg of either the proteolytically inactive passenger domain (EatApH134R), EtpA, or both antigens on days 0, 14, 28. Colonization of mice following immunization with the adjuvant alone compared with single and dual antigen vaccination. Comparisons between groups were by Mann Whitney two tailed nonparametric testing. (One mouse died during the vaccination period in the LT/EtpA group and was therefore excluded from the analysis). For mice with no detectable colonies following challenge, the number of cfu is arbitrarily reported as 1 (10°) cfu, the theoretical limit of detection.
Enterotoxigenic Escherichia coli remain one of the most common causes of infectious diarrhea worldwide, and severe disease caused by these pathogens persists as leading cause of death among young children in developing countries. Despite recognition of these toxin producing E. coli as a cause of severe cholera-like diarrheal illness more than forty years ago, there remains no effective broadly protective vaccine for ETEC.
Most vaccinology efforts to date have focused almost exclusively on a subset of plasmid-encoded antigens, namely the colonization factors (CFs) and heat-labile toxin. Vaccines based on this strategy have faced several impediments. First, the CFs are quite diverse with more than 26 distinct antigens described to date. In addition, a number of recent vaccine studies have suggested that simply engendering immune responses to CFs and/or heat-labile toxin may not be sufficient to provide sustained broad-based protection[14–16].
Recent studies of ETEC pathogenesis suggest that a number of virulence factors in addition to the CFs are involved in efficient delivery of toxins to their cognate receptors on the epithelial surface[2,22,29,30,48,58]. Similarly, the immune response to ETEC infection appears to involve many proteins[25,47] in addition to the classical antigens that are the present focus of most vaccines. Collectively, these findings suggest that there may be additional molecules that could be targeted to interdict toxin delivery by these pathogens, expand the list of potential protective antigens, and complement existing approaches to vaccine development for ETEC[59,60].
A major challenge to ETEC vaccine development in general is that the most highly conserved antigens of ETEC, typically encoded on core regions of the chromosome, are also shared with commensal E. coli. Included among these chromosomally encoded conserved proteins are two antigens studied here, YghJ and EaeH that were recently shown to be important for ETEC virulence. While the present studies also demonstrate that these proteins are recognized during the course of ETEC infection, the degree to which these antigens can be safely targeted in vaccines without inadvertent disruption of the intestinal microflora remains to be studied.
The inherent plasticity of E. coli genomes contributes substantially to the difficulty in defining antigens unique to the ETEC pathovar that are widely conserved. No single antigen exclusive to these pathogens, but universally conserved in this pathovar, has been described to date. Some have suggested that this might be predicted based on the fact that the plasmid-encoded heat-labile and/or heat-stable toxins, which define the ETEC pathovar, could form a minimal complement of virulence genes in wide variety of E. coli host strains. Nevertheless, earlier studies conducted on phlyogenicaly disparate strains from Guinea Bissau and Chile suggested that genes encoding two pathogen-specific antigens EatA and EtpA were present in a majority of strains.
In this context, we examined the gene conservation and the actual production of these proteins in a large collection of well-characterized strains from Bangladesh, complemented by strains from other locations that were associated with severe disease and for which there were available clinical metadata. Notably, two plasmid-encoded ETEC pathotype-specific antigens, the EatA serine protease and the secreted EtpA adhesin molecule were shared broadly among strains belonging to different CF groups with the exception of strains that produced CFA/IV antigens CS4, CS5, CS6 which only infrequently produced EtpA.
The studies reported here represent the largest screen for EtpA and EatA secretion in ETEC performed to date. Earlier studies reporting that genes encoding both proteins were highly conserved relied on either PCR or screening of draft genomes for the presence of the corresponding loci. In general, we found high degrees of concordance between the presence of these genes by PCR and production of the corresponding protein. We should point out however that draft genome assemblies typically fail to encompass the entire etpA gene as automated assembly algorithms cannot faithfully incorporate the multiple repeat regions comprising two thirds of etpA. This could impact interpretation of gene prevalence in ongoing large-scale ETEC genome sequencing projects. The prevalence of EtpA and EatA (56 and 59%, respectively) as determined by examination of protein expression in our study was slightly lower than previously reported in earlier studies that analyzed strains from Guinea Bissau, where both genes were present in 75% of strains ; or Chile, where etpA and eatA, were present in 71 and 75% of strains, respectively. Nevertheless similar to these earlier studies, the strains that produced these antigens belonged to many different phylogenies suggesting that genes encoding these antigens have been widely dispersed.
The analyses of strains in these studies largely focused on isolates from Bangladesh. However, these data are potentially relevant for vaccine development for a number of reasons. First, Bangladesh is highly endemic for enterotoxigenic E. coli infections, and consequently remains an important site for vaccine field trials. In addition, ETEC has been under study in this region since the discovery of this pathotype, permitting us to compare sequence variation in candidate antigens over four decades. Understanding both current prevalence and sequence conservation of potential novel vaccine antigens in this population over time will be particularly important for making rational decisions about their inclusion in future iterations of ETEC vaccines. Finally, the geographic and temporal dispersal of genes encoding EtpA and EatA in multiple phylogenic backgrounds, further attests to importance of studying these molecules as potential vaccine targets as previously suggested by others[37,49].
The optimal formulation of an ETEC vaccine has yet to be defined, and many questions pertaining to the nature of protective immunity that develops following infections with these pathogens remain. Nevertheless, the data presented here do suggest that the novel pathovar-specific antigens could complement existing strategies for ETEC vaccine development by broadening the antigenic valency. Whether the expanded coverage afforded by inclusion of additional pathotype specific antigens would enhance vaccine efficacy beyond that presently achieved by targeting CFs and LT will need to be determined empirically.
S1 Fig. EatA passenger domain alignments of predicted EatA sequences corresponding to multiple phylogenies from strains belonging to disparate geographic origins.
The conserved catalytic triad at amino acids H78, D106, and S211 is highlighted by gray background shading. Geographic origin of strains is depicted in the color key at left of the alignment. Alignments were performed using CLUSTAL Omega (release 1.2.0 AndreaGiacomo)  algorithm plugin for CLC Main Workbench.
S2 Fig. EtpA alignments of predicted sequences corresponding to multiple phylogenies from strains belonging to disparate geographic origins.
Geographic origin of strains is depicted in the color key at left of the alignment. Alignments were performed using sequence alignment algorithm of CLC Main Workbench v6.9.1 with the following parameters: gap open cost = 10.0; gap extension cost = 1.0; end gap cost = as any other; alignment mode = very accurate (slow); redo alignments = no; use fixpoints = no.
S3 Fig. Immune response to selected ETEC proteins in infected patients and Bangledeshi and US controls.
Shown are (IgG) kinetic ELISA responses (in Vmax, milli-units/min) to recombinant proteins comparing convalescent plasma from patients hospitalized with acute ETEC infections at the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh with controls (c) from Bangledeshi adults, and children, as well as plasma from age-matched children from Saint Louis Children’s Hospital (slch). Antigens included two plasmid-encoded ETEC specific antigens (a) EtpA, and (b) the EatA passenger domain; and two chromosomally-encoded conserved antigens (c) YghJ, and (d) EaeH. All plasma samples were diluted 1:4096.
S4 Fig. Immune responses to EtpA following volunteer challenge with ETEC H10407.
All sera were diluted 1:4096 prior to testing against rEtpA-myc-6His followed by detection of total antibody (IgM,IgG,IgA) in kinetic ELISA. Pre and post values (open and closed circles, respectively) represent collective data from 2 independent ETEC H10407 challenge studies CIR218 and CIR193a. Data from CIR218 are shown as pre-challenge (d-2, open blue circles) and (d28, closed blue circles), while data from CIR193a appear as open grey circles (pre-challenge, d0) and closed grey circles (post challenge, d9). Dashed horizontal lines represent geometric means. P value represents comparison of pre and post-challenge samples by Mann Whitney 2-tailed analysis.
S1 Table. Oligonucleotide primers used in this study.
Shown in the table are oligonucleotide pairs, and predicted product sizes for ETEC pathotype-specific virulence gene amplification in these studies.
S1 Dataset. S1 Dataset.xlsx contains the complete listing of all 181 strains analyzed for production of the secreted EtpA, EatA antigens (tab 1), and YghJ (tab 2).
Tab 3 shows 91 unique isolates sequenced by the Genome Sequencing Center for Infectious Diseases (GSCID) which were screened for the presence of the eaeH gene by BLASTP homology searches. Note that in the dataset, disease severity is classified numerically (0 = isolate from asymptomatic colonization, 1 = mild disease, 2 = severe cholera-like illness). The presence or absence of a given protein or gene in each strain is defined in a binary fashion where 1 = yes; 0 = no.
Conceived and designed the experiments: JMF FQ DAR QL. Performed the experiments: QL RK AS. Analyzed the data: QL DAR JF. Contributed reagents/materials/analysis tools: FQ DAR. Wrote the paper: QL JF DAR.
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Four Zimbabwe national cricket team players have positioned themselves to win places at this year’s Indian Premier League (IPL) by registering for next week’s auction.
BY Sports Reporter
Registration closed on Tuesday and it was reported by Indian media that five Zimbabwean players had registered, but there are only four local names on the list.
Batsmen Malcolm Waller, Sikandar Raza, Solomon Mire, leg spinner Graeme Cremer have all put themselves up for selection.
A total of 232 foreign players have signed up for the auction where there are also 736 Indians who will all be fighting for the available 70 slots.
The IPL franchises now have until Monday to submit their shortlist of players that will make up the final auction list.
Zimbabwe’s quartet will be looking to emulate Ray Price, Tatenda Taibu and Sean Ervine, who have all played at the lucrative T20 jamboree.
Raza, who has played in different T20 leagues this year, will be hoping that this would be his breakthrough year, having enjoyed good form in the limited overs cricket for the national team.
Cremer, who underwent surgery, will also be looking to bounce back, having also played well for the national team during the International Cricket Council World Cup qualifiers that were held in the country earlier this year.
Over the years, Zimbabwean players have generally been over-looked by the IPL teams and with the challenges faced by the national team, their chances have continued to dwindle.

domain: news


text:
In the rigmarole of providing caregiving to all and sundry, women forget to care about themselves, and in not saying no to the world, they keep saying no to themselves. Saying no is the first step to self-care and for most women, self-care is akin to a luxury or indulgence. Poet and essayist Audre Lord had written in her 1988 essay, “A Burst of Light”, soon after she was diagnosed with cancer for the second time: “I had to examine, in my dreams as well as in my immune function tests, the devastating effects of overextension. Overextending myself is not stretching myself. I had to accept how difficult it is to monitor the difference. Necessary for me to cut down on sugar. Crucial. Physically. Psychically. Caring for myself is not self-indulgence, it is self-preservation, and that is an act of political warfare.”
Self-care is merely taking back control of our lives from a culture that subverts women. It is about women’s physical and mental well-being and hence, their basic human rights, and overturning a world that runs on the exploitation of their invisible labour and caregiving. Self-care is an important act of rebellion and it starts with saying no.
Research suggests that women may feel the need to prioritise the needs of others over their own, leading to difficulty in declining requests without feeling guilty or stressed about the consequences. Not being able to say no and not setting boundaries leads to increased stress and anxiety. Individuals who struggle with boundary-setting often report high levels of stress and anxiety. American Psychological Association’s Gender and Stress reports have consistently found that men and women react differently to stress. Their 2012 report found that women reported symptoms of stress as lying awake at night, overeating or eating junk food or completely skipping meals. They also reported other symptoms of stress such as “feeling depressed or sad to experiencing headaches and changes in sleeping habits” or constant fatigue, anxiety, feeling overwhelmed, feeling exhausted, feeling like crying, or suffering from upset stomach or indigestion.
All of these findings are so important even if they are not in the Indian context. For years, I have struggled to understand these symptoms in myself. Women I know, friends and acquaintances, struggle with it too. And yet, we push it under the carpet and carry on. We try to manage our stress but more often than not, we fail. I have failed spectacularly to manage my stress. If you eavesdrop on women’s conversations, I can guarantee you that you will hear one phrase across the board – “I am so exhausted.”
According to the APA, while women were more likely to report physical symptoms as effects of stress, they were also better at devising stress management strategies. Better at devising stress management strategies or shoving it all under the carpet?
For the better part of my life, I thought I was successfully controlling my stress but I was merely devising ways to bypass it without addressing it because if I was really devising strategies, it would have started with saying no to unreasonable demands on my time, not just at home or work but to so many other people I know or barely know.
The APA report also found that women were much more likely than men to say that having a good relationship with their families is important to them, but fewer women say they are doing a good job at succeeding in this area; they are also more likely than men to say that having a good relationship with their friends is important to them, even though friendship is cited less often than family for both men and women; and nearly half of all women said they have lain awake at night in the past month because of stress.
Women are apparently also more likely than men to say that they lacked the willpower to make lifestyle and behavioural changes, and that they needed to be less fatigued and have more confidence in themselves to improve their willpower. And finally, interestingly, while most men would say they needed to earn more money, women said they needed to find more time. Yes, more time. Sometimes, I wish days had 48 hours instead of just 24. Or that I could be less tired so I could make use of more of those hours, but since I have hit 40, I can barely stay up beyond 10 pm. I have also had Covid-19 four times, after which I feel like exhaustion is the only constant in my life. And in a cruel twist of fate, I can’t sleep beyond 4 am because anxiety about losing hours of sleep is ever-present in my mind.
If I could say “no” more, I would probably have fewer demands on my time. And believe me, every day, I practice various ways of saying no – not hesitantly but determinedly, confidently and without guilt peppering my words. Just the other day, a woman acquaintance apologised to me for saying a “blunt no”. They went on to explain that they had so much demand on their time that they were struggling to cope. I messaged them back saying “I love a blunt no” and in that moment, we were united because we realised the power of saying no. I respected her more for saying a blunt no, but I also recognised the societal conditioning that makes us apologise for saying a blunt no. And since then I have said a few blunt noes of my own, it feels good. But the fact that I still struggle with saying no means that I am always burnt out – professionally and personally.
And while we might think that burnout is specifically a work-related problem, I believe that it is not. Women suffer personal as well as professional burnout that usually manifests in overwhelming exhaustion, feelings of cynicism and detachment from the job, and a sense of ineffectiveness and lack of accomplishment. Sound familiar? To me, it does, and I felt it keenly in both my personal and professional life. If a workplace is defined as any place where a person works, then that makes the home a woman’s workplace too. Hence, would it be so illogical to assume that like we face burnout in the workplace, we face similar burnout in our homes too? When the two collide and combine for me, it sometimes manifests in physical illnesses, sometimes in an out-of-body feeling where I feel like I am merely following the motions of my life, and sometimes in just vacant thoughts. And of course, there is anger, resentment and depression.
As Paulo Coelho said, “When you say ‘yes’ to others, make sure you’re not saying' ‘no’ to yourself.” A 2021 article, “How Women Who Do It All Can Combat Burnout”, examined how women who were used to doing it all could beat burnout by saying no. “If you say ‘yes’ more to others then you’re saying ‘no’ to yourself. Learn to re-evaluate your responsibilities and become OK with letting things go. If you’re maxed out at work, then consciously let something else go,” the article had advised. “At home, re-evaluate your to-do list and consider outsourcing some of the house tasks to your partner or hiring a service. Remember, ‘no’ is a complete sentence in and of itself. You don’t need to qualify your answer or give any rationale.”
Excerpted with permission from How Not to Be a Superwoman: A Handbook For Women to Survive the Patriarchy, Nilanjana Bhowmick, Penguin India.

domain: literature


text:
TechSpot means tech analysis and advice you can trust.
As Google moves forward with their acquisition of DoubleClick, they have faced a few setbacks as various regulatory agencies have been probing the deal. Most recently, the EU has decided to further extend their investigation into the deal to see if it would potentially stifle competition. While Google was hoping to close the deal before the end of the year, the EU now says they will not come to a decision until April of 2008.
Obviously this has upset Google, and it may upset them further if the EU decides that the acquisition requires "modification" to be approved. It has already received the go-ahead in the U.S., so Google still has options there.
Microsoft is still vehemently opposed to the deal, despite them also taking similar measures with their purchase of aQuantive.

domain: economics


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Bubble Bubble. Bubble Bubble 2. Caspers Mystery Mirror. Caught in the Widow's Web. Charms and Witches. Count Spectacular. Crazy Halloween. Crazy New Year.
Creatures of the Night. Creepy Carnival. Creepy Castle. Creepy Cuddlers. Crypt Keeper. Curse of the Werewolf Megaways.
Dancin' Zombies. Dangerous Beauty. Dark Carnivale. Devil's Delight. Devil's Heat. Devil's Number.
Dia de los Muertos. Dia De Muertos. Diablo Diablo Reels. Dracula Riches. Dracula's Blood Bank. Dracula's Castle. Dracula's Family. Ducks Till Dawn.
Dungeon Quest. Esqueleto Mariachi. Fairground Fortunes Ghost Train. Fear the Zombies. Fortunes of the Dead.
Frank's Freak Spins. Frankenslot's Monster. Freaky Frankenstein. Freemasons Fortunes. Bounty Haunters 0. Fugaso Horror Castle 0. Vampire Princess of Darkness 0.
Fiesta de la Memoria 0. Pumpkin Smash 0. Maji Wilds 0. Book of Eon 4. Lucky Halloween 0. Ellas Riches 0. Transylvanian Beauty 0.
Halloween Fruits 3. Halloween Toons 0. RTG Witchs Brew 0. Halloween Fortune II 0.

domain: game


Citi Bike expansion into Sunset Park, Windsor Terrace, and Greenwood Heights is underway, NYC DOT announced today. The project is part of a 93 docking station expansion and will extend the bike’s services from 15th Street to 67th Street and from the southwest corner of Prospect Park to Church Avenue.
The bikes and docking stations are set to all be installed over the next two to three weeks. So far, about half have been completed. According to Citi Bike, this year has been the single largest growth year for CitiBike in terms of service expansion. As the name of the expansion suggests, 93 CitiBike stations will be installed in Brooklyn as part of this growth.
Carlos Menchaca, the City Council Member for District 38, said he was “pleased this is finally happening” after pushing for Citi Bike docks in Sunset Park. Menchaca, who is currently a candidate for this year’s Mayoral race, has long been a proponent of safe, bike-friendly streets.
In the past, bike expansion has been controversial in Southern Brooklyn, as Community Board 10 members were concerned about traffic patterns and bus station interference.
Citi Bike hit 100 million all-time rides last year, with over 42.1 million miles pedaled in the last year alone. The pandemic brought out a new community of bike riders, surging business and creating huge lines at neighborhood bike shops. Ridership of Citi Bikes was higher than ever from last September-December with more than 600,000 first-time riders for the year.
The Executive Director of Transportation Alternatives, Danny Harris, said that “prioritizing people over cars” was essential to the group’s 25×25 report, an initiative that advocates giving one-quarter of the space allotted for cars to space for people by 2025, and that this expansion was a step in that direction.
“One of the best ways to do so is by bringing bike sharing to more communities. At the same time, we must also grow the protected bike lane network, and Transportation Alternatives will continue to work with the de Blasio administration and local communities to ensure everyone has safe streets,” Harris said.
The growth is part of Citi Bike’s Phase 3 expansion, set to be completed in 2023, which will double the footprint of the company. Manhattan and the Bronx are next on the list for service expansions.


Natrona Comes Together Association Breaks Ground on New Playground
The folks of the Natrona Comes Together Association spearhead progress and improvements of their small community in HarrisonTownship. The organization secured grant monies to demolish more than 15 dilapidated properties that pose as hazards to District children. It has hosted numerous events, including a National Police Night Out, in hopes of connecting residents to their community.
This Friday, Oct. 3, Dr. Karol Galcik and several elementary students will attend a groundbreaking of a new playground along River Avenue. Among the improvements, according to the association, will be a state-of-the-art children's playground, glass block walls composing a theater space, updated basketball courts, and other amenities. The association also solicited young artists to assist in designing murals and other art in the playground area. The modernized area will serve as a community venue for upcoming events and programs the association plans on hosting.
This has been two years in the making, said Bill Godfrey, president of NCTA. We are so excited to be able to provide this kind of town center for our children and our community.
Activities begin at 10 a.m. with events for children, including storytelling, puppets and balloon sculptors, as well as a luncheon for all who attend. The groundbreaking will begin at 11 a.m.
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Watch out for revenue when purchasing Cordella Chaise Lounge By Willa Arlo Interiors. More often than not furniture is placed on purchase. If you require a specific piece, it will save you a ton of money by patiently expecting the retail price to become lowered. You may ask your preferred Cordella Chaise Lounge By Willa Arlo Interiors store if the product will be noted for less money. If you require a new furniture, try out finding one particular with many completely covered pillows. These pillows stay longer and might be flipped on a regular basis. Essentially, you should obtain a furniture with easily-removed handles so that you can very easily clean them. Removable includes can be substituted very easily in case you are not able to thoroughly clean a blemish Acquire Cordella Chaise Lounge By Willa Arlo Interiors in the future around. At the end of this coming year, it is actually common for stores to get these items away from their display room flooring. This can result in some great sales and savings.
Top Best Chaise Lounge Chairs Reviews
Probably the most useful equipment of the home is the stool. Bar stools are useful wherever it is required it may be in the kitchen area or for other gatherings in your own home. Barstools could be operated manually that is simple in functioning. These barstools can be modified manually to the level of enhanced comfort zone. There are various types of club feces which is made of wooden and metals, for this some types of chair there may not have a flexible tool which might hot lower the peak from the seat but rather developed in a well sited manner. Bar stools adds extra kitchen and eating seats with fashionable appearance. It retains guests lingering of the contemporary home. Finding bar chair with appropriate the perception of home is the challenging 1. You need to find the correct style with correct price and quality of the bar stools to be bought. We have to discover the ideal height from the stools that suits our height, and must find the main difference between your club feces and counter feces. Finally, you have to find stools at the correct price, correct high quality with guarantee with required sturdiness which must satisfy us for the spending.
1.Gowans Chaise Lounge By Three Posts
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This ultra-elegant, higher-sovereign sofa is designed to support cutting-edge living room innovation. It's created as a 2-item sectional couch combined with a beautiful chair. It also provides cellular arm rests and beneficial back support. The gorgeous tapestry is completed with darkish false leather, securely tufted. You are able to feel comfortable padding with foam within the seat cushions. This guarantees a delicate sensation when you rest following a boring day. The sofa is strengthened with wooden edge engrossed in dark artificial leather. False tufted leather and powerful pinus radiata give severe glare. It is true that it requires another appear since it is more costly of computer really appears to be. Talking about dimension, its large! This is exactly what adds more superstars to this set. The peak can also be impressive for high and brief people.
2.Richards Relax Chaise Lounge By Latitude Run
Read more about Richards Relax Chaise Lounge By Latitude Run
When it comes to purchasing room furniture, leather-based is definitely a good option. It doesn't only look great with most designs, but its extremely long lasting (its the ideal materials for a home with kids or animals) and its extremely-simple to clean, too. The downside of leather furniture? It may have a higher cost than fabric, microfiber or faux leather Richards Relax Chaise Lounge. This reclining loveseat stays a financial budget-friendly choice because of one genius technique: its sitting area is upholstered with leather, as the sides are padded with more inexpensive faux leather-based. That means you get the appear and feel of a full leather loveseat without the hefty price tag. Make use of the lever around the loveseats arm to kick back and take it easy on its higher-density froth filling up, and consider all the money you saved. This specific loveseat posseses an additional reward: professional assembly is available in numerous locations of the country for an additional fee.
3.Verona Chaise Lounge By Three Posts
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Even though this lying loveseat has a heftier cost, its classic style, options for personalization and automated reclining system allow it to be well worth the dough. Its handcrafted wood body is cushioned with lower blend soft cushions for a softer feel than you would get from high-denseness foam soft cushions, and also the furniture will come in a whopping 72 color and material combos. The supply of so many options makes it extra easy to set this lying loveseat with different patterns, materials, highlights and decorations styles. Plus, if you have children or animals (or each!), you can select a stronger, stain-resistant material. One client shared the Sunbrella canvas material has organized nicely against her children and pets. This lying loveseat could have a classic, aged-college look, nevertheless its constructed-in technology is anything but. Not only does it have an automatic reclining program (that provides you with a full recline, together with your foot rest totally parallel to the seat), but it features a USB port for implementing or just getting your iPad, iPhone, Amazon kindle or laptop.
4.Auberge Linen Chaise Lounge By Canora Grey
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This established accompanies the entire utility of Person Sofa by having an excellent couch design. It includes restricted hands, firm seat soft cushions and full-dimension sleeping pad with inner springs. It is eliminated beneath to wash it and remove it with a easy lifting device. This sleeping pad is strengthened having a durable metal edge. is really a measuring guideline and has a Bisexual-falling apart emphasize. One of the deserving items to point out is the convenience! Place this super comfy sofa inside your comfy family room. Apart from providing an all natural and stylish atmosphere, it provides a beautiful personality for your visitors. You can make your buddies remain for any enjoyable night of films. The couch has carcasses with blocked edges and fake-finishing on its thighs. The cushions are, nevertheless, adjusted and include great flexibility. It is wrapped having a thick polyethylene dietary fiber with a drawstring edge.
5.Pavilion Leather Chaise Lounge By Fine Mod Imports
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This set is upholstered in a push and comfy distinctive padded purple velvet. This gives it the amazing comfort that it is cherished for. Again it facilitates chaise design seats for adequate comfort. Furthermore, the sinus springtime foundation adds to the comfort and durability. Additionally, the couch has hard wood frames that add to its toughness and durability. The couch has soft cushions on both the seat and the back again. But still, the sofa can move from a seated position to some reclined placement easily. That said, simply to repeat, the sofa is very gentle and provides exceptional comfort and ease.
6.Cedarville Chaise Lounge and Ottoman Set By Three Posts
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This plush group of city-chic sofas is created to supply an adorned domain. Actually, even your modest areas may have you required to have an immaculate film night. Lay him down to get a completely altered bed. This avant-garde sleeper futon mattress is accessible in various shades. This includes some bright shades unmanageable for example Orange, Mild Blue, and lightweight Purple. Place them in your innovative studies or attic to assess the exuberance. It is also available in decreasing shades for example dark, darkish and darkish. These subtleties, once more, print a respectable aspect of the higher class. With the splitting element of your back, you are able to rest for your preferred position. This sofa appears wealthy with tufted material consistency upholstery. The good thing is that it is personalized with hypoallergenic filling. A conclusive outcome provides a comfortable rest.
7.Abreu Chaise Lounge By Willa Arlo Interiors
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Whilst the back of this couch hints at the Chesterfield, using its rows of tufting, a tux couch has cleaner, much more angular outlines. It is said to have been the bellwether of more modern designs in the 1920s. While some say this couch style takes a title from Tux Recreation area in Ny, it is also considered called after the traditional fancy mens match. You can determine a tux sofa by its hands that are the same height because the back. The tufting on the back of the sofa and its rectangle-shaped silhouette will also be traditional qualities. Soft cushions add comfort and ease for this high-equipped style. Also, as the couch over from Upcountry is padded in leather-based, this popular style is often completed in textiles of all kinds, including the very trendy velvet.
8.Cynthia Chaise Lounge By Lark Manor
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With almost 100 5-celebrity reviews, this lying loveseat is recommended for its comfort and ease, sturdiness, appearance and ease of assembly. In addition to soft, but supportive soft cushions, it provides a sliding system, so you can place your ft up and rock (though, not at the same time.) Padded with extremely-long lasting glued leather-based, this lying loveseat will avoid holes, rips and stainswhich is a good factor, since it also includes a storage compartment system (where you can conceal your snacks, of course) along with a two-mug holder. Regardless of whether you are deciding set for a Blockbuster online excessive or viewing football together with your team, this lying loveseat provides the durability and comfort without emptying your wallet.
9.Park Chaise Lounge By Klaussner Furniture
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The Lawson might be called quintessential United states sofa style. It's comfy and simple: The boxy shape is large and usually has 3 back soft cushions in addition to three seat soft cushions. The classic Lawson also offers A taller back and box-formed cushions which have welts in the edges, as do the back pillows. Ideal for snuggling and sleeping, this sofa style was designed for Jones Lawson, an American copper tycoon in the turn from the century. He desired a sofa which was very different from the picky Victorian styles which were typical at that time. Present versions of the Lawson can also incorporate metal or wood contained in the arms. Most importantly, this couch style is the one that will morph its feel using the fabric you select for the upholstery. The form is really flexible that it may be attractive, magnificent, commercial, informal or formal depending on your fabric option.
10.Cass Chaise Lounge By Ebern Designs
Read more about Cass Chaise Lounge By Ebern Designs
This fits you if all you need is an attractive coach. It's designed with a flared body and pillow top amrests in a cool cobblestone grey. Now, it's resilient froth cushions that are wrapped up in in a polyester furniture to bring about the specified comfort and ease. It has a durable part obstructed frame that adds to the sturdiness. Also, the feet are in a fake wood finish. We have an impressive gray color that matches with any decor. Its dimensions are 89 T by 39 D x 40 They would therefore big enough to support you along with your family/buddies. More importantly, it occurs fully put together. This saves the agony of getting to assemble the established.

domain: design


i’m new at sewing and have been using a twin needle to sew stretchy jersey/knit fabrics. everything is turning out great, plenty of ease, perfect fit BUT my stitches still show on the outside. Initially i was using a single needle and a zig zag stitch but that was showing so i figured it was the zig zag’s fault, now i’m using a twin needle and they’re STILL showing. the shirt is loose for me but when i pull it over my head/shoudlers, it stretches and i guess that’s why it’s pulling at the seams. Anyway, i don’t want the stitches to show.
what can i use to stop them from showing?
since it’s just side and sleeve seams, could i just use a straight stitch with a single needle ?
or is that a no-no on jerseys/knit fabric?
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What about the new Continental Edison CELED50SA20B3 ?
A good mid-range television. This is how we can introduce the Continental Edison CELED50SA20B3.
Concerning technology, it is worth noting that this model is delivered with a Ultra HD 4K resolution. With a diagonal of 49.5 inches, this mid-range model canbe considered as a rather cheap television .
Although placed in the middle of the range, the manufacturer has tried to offer the viewer an image quality that allows an immersion.
We will see in detail if their objective is reached.
The design of the Continental Edison CELED50SA20B3
The Continental Edison CELED50SA20B3 with its simple design is an interesting alternative for anyone who wants an elegant television set in the mid-range segment.
A good overall score for this TV; it corresponds to design in the spirit of the times. A quality of materials and a visual aspect that does not make you insensitive.
A mid-range television set that relies heavily on its design and the quality of its materials; arguments that Continental Edison has been promoting for some time now on these products. All this for a price that stays contained.
TV screen presentation Continental Edison CELED50SA20B3
The Continental Edison has a correct and well-calibrated image quality, but you have to do with a limited native contrast that is suitable for daytime viewing; forget about home theater programs in the dark.
With this new model, Continental Edison confirms one of its technological choices.
The Continental Edison television set displays a Ultra HD 4K resolution. At the same time, we are dealing with a nice display on the screen.
With a frequency of 50 Hz, you can forget about using this TV in game or home theater mode. image shifts can be important and the accuracy (compared to the original source) is very average.
Note that with advanced technology, this TV gives you a Ultra HD 4K quality picture .
In conclusion on the display, with the CELED50SA20B3 Continental Edison makes the choice of the mid-range. No more.
The sound of the Continental Edison CELED50SA20B3
A brief listening allows us to see that it does not work miracles. The sound is far from exceptional. That’s enough for TV shows, but not for home theater showings.
The television set is equipped with two loudspeakers and a power output of 10 W.
The sound of this TV is in a word immersive, and you feel like being in a small movie theater.
The sound quality of this TV is average. Don’t push the volume too hard, you’d be disappointed.
The Intelligent aspect of the Continental Edison CELED50SA20B3
For the software part, the CELED50SA20B3 Continental Edison has an operating system Android TV. You have the opportunity to explore the world of entertainment.
With its Wifi or network connection (local LAN), this TV gives you the possibility to download apps or movies directly. An interesting option in the age of the cloud and online games.
CELED50SA20B3 from Continental Edison – Its connectivity
This television Continental Edison CELED50SA20B3 offers a rather classical connectivity : mixed inputs HDMI, the ports as well as the audio are available on this model.
Whether it’s your external hard drive, NAS or game console, all these elements can be connected to this TV.
It is with 8 possible connections that this CELED50SA20B3 is presented. Almost all the devices in your home can be connected: the box, the game console, a multimedia server, and many other devices, in addition to your DVD or Blu-ray player.
In the end, this TV provides you with the following features:
2 USB ports, 4 HDMI ports, 1 audio output, 1 component plug (Video)
Technical characteristics of the Continental Edison CELED50SA20B3
Here is a summary of the technical characteristics of the television set:
|Sizes of the television without stand||85|
|Screen size in inches||49.5|
|Screen resolution||Ultra HD 4K|
|Tuners available||DVB-T DVB-T2 DVB-C|
|Number of tuners||3|
|Number of USB ports||2|
|Number of HDMI ports||4|
|Number of audio outputs||1|
|Composite Input (Video)||Yes|
|HDR Dolby Vision Compatible||Yes|
CELED50SA20B3 from Continental Edison – The conclusion
Finally, this new Continental Edison CELED50SA20B3 is a television set that can go anywhere; it can meet the majority of needs.
For the display technology we are in 4K; you have a very good quality image, and an impressive sound quality; with its well thought out aesthetics, the result gives you a device that you want to buy.
If there are any positive points to remember, here are 3 interesting ones:
the display frequency, the sound power, the depth of the colours.


Biden was visiting the state to pitch his deeply unpopular $2.5 trillion infrastructure plan.
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As he arrived, thousands of supporters waved their “F**k Joe Biden” flags and gave POTUS the middle finger as the presidential motorcade drove by.
After arriving, Biden spoke to a tiny crowd of people at the training facility for the International Union of Operating Engineers.
He was clearly triggered by the protests and bizarrely commented that the “F**k Joe Biden” pandemic spreading across America was why he got 81 million votes.
“And not withstanding some of the signs that I saw coming… that’s why 81 million Americans voted for me. The largest number of votes in American history,” he told the tiny group.
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- Hours 11:00am - 10:00pm
- ETA 45 - 60 min
- Delivery Minimum $15.00
- Delivery Cost $1.00
Restaurant is not currently accepting online orders.
It will reopen tomorrow at 11:00am.
One coupon per order.
All salads have iceberg lettuce, tomatoes, onion, green pepper & cucumbers.
Served with white or red sauce.
Served with red sauce.
Served with White sauce.
Served with Special sauce.
Served with homemade ranch dressing or blue cheese dressing & your choice of sauce.
8 Individual pieces.
Mustard on request.
All personal pasta dinners come with a small tossed salad & garlic bread. All family size pasta dinners come with pieces of garlic bread, serves 4.
Served with homemade ranch dressing or blue cheese dressing.

domain: food


Oxfam, Etherisc and Aon team up to launch blockchain-based agricultural insurance policies. With farmers risking to lose their crops due to bad weather conditions, the deveolped system initiated payments for those affected. The system still has room for improvement which the companies plan to take care of.
“We are proud to have real-world, on-the-ground success from a blockchain solution for microinsurance,” Michiel Berende, the Chief Inclusive Officer at Etherisc, said. “We are delighted with the first phase results and we are excited to drive on and help more farmers” according to a source from PRNewsWire.
The effort is pulled by three companies in different industries. Oxfam; an organization working to reduce poverty, Etherisc; a FinTech in the insurance industry and Aon; leading services firm offering healthcare solutions.
Farmers struggled to acquire insurance policies due to high costs, a lack of understanding and when claims can be paid. Blockchain addresses those issues by automating actions. Claims will be paid automatically and administration costs will drop significantly.
Even though many of the issues with traditional insurance policies were fixed, others arose: lack of electronic devices and internet access, absence of a network and the farmers’


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Ryan and Shannon’s wedding at the Flower Farm
I am so super duper excited to post this wedding! I have to start off by saying that Ryan and Shannon are two of the sweetest, friendliest, energetic, completely-full-of-personality people you will meet. From the moment we met them, we knew they would be a blast to get to know and we were right! We had SO much fun with them on their engagement session incorporating all of their ideas into the photos. Fast forward to their wedding day and I could not imagine anything more perfectly “them”. The weather that day was perfect at the Flower Farm in Loomis, which is one incredible venue. I don’t think I can get enough of this place. The photo opportunities are nearly endless. Everywhere you turn there is something beautiful. Pair that with a gorgeous bride and handsome groom and we were in photographer’s heaven!
And then there is Shannon. I could not stop taking photos of this gorgeous girl! She had so much joy bubbling up inside her that you could just see the happiness in every photo. Don’t even get me started on little Tucker! I mean, a bride and a puppy? How much cuter can you be?!
While I was basking in the loveliness of Shannon and her best girls, Geoff had the honor of photographing Ryan. Now here is a guy every dad wants his daughter to marry — so sweet, so thoughtful, funny, and so cute! I said it before, but these two are simply perfect for each other.
Because photography was so important to Ryan and Shannon (and ’cause they are just plain awesome) they decided on a First Look. If you are not familiar with a First Look, basically, prior to the ceremony, we set the bride and groom in a beautiful location and they get to see each other for the first time, just the two of them, and get to enjoy some intimate alone time (probably all they will get all day!) while Geoff and I snap away. It takes some of the edge off on your wedding day and allows us to get time for some amazing photography. Case in point, Ryan and Shannon.
Who does not love a candy bar!? I always love those pops of bright colors! I also adore the way they used one of their engagement session photo for the sign. SO glad we stopped at that candy store in Old Sacramento!
Their cake, made by Cakes by Jami was so simple and so beautiful.
I cannot say it enough, Ryan and Shannon THANK YOU for choosing us to capture your wedding day. Geoff and I feel honored just to KNOW you let alone have our photographs be a part of your lives. We know you are going to have a long happy marriage and we wish you nothing but the best for the future! Our only hope is that we can have as much fun in our own marriage as you two seem to have around each other.
Ceremony and Reception Venue: The Flower Farm
Officiant: Reverend Paul
Florist: Bel Air Florist
Catering: Brown Brothers Catering
Cake: Cakes by Jami
DJ/Entertainment: Mix Masters Pro
Bridal Hair/Makeup: Vibes Salon

domain: artistic


As the UK’s largest union of financial professionals Unite is launching an investigation into the pressure selling culture within Britain’s banks and insurance firms. How does this impact you and your colleagues?
Fill in the short survey below and make sure the views of your workplace are heard. All information received will be treated with strict confidence. For more information contact Ben Norman.