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Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection).
IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has become endemic in Africa, Asia, and the Americas and has been deemed a global threat by the World Health Organization (1, 2). ZIKV infection during pregnancy is causally linked to severe neurodevelopmental damage, including fetal microcephaly, intracranial calcifications, neurodevelopmental damage, and ocular anomalies (3). In adults, ZIKV may also trigger Guillain-Barré syndrome (4–6). In urban and suburban environments, ZIKV is transmitted in a human-mosquito-human transmission cycle. Transmission is also reported in sexual partners (7, 8) and via blood transfusion (9). Molecular assays for detection of ZIKV gene products in body fluids are the gold standard for definitive diagnosis of active infections (1, 10). However, the median duration of viral RNA is 22 days in whole blood and 10 days in plasma (11). Furthermore, many infections are asymptomatic, associated with only mild acute disease, or occur in areas where access to medical care is limited and blood collection is unlikely to be achieved during the acute phase of infection. Thus, teratological risk assessment during pregnancy and infection incidence and prevalence surveys also require serology for detection of gestational infections after resolution of viremia (12).
Cross-reactivity between ZIKV and other flaviviruses may complicate differential serodiagnosis and efforts to investigate the epidemiology of infection and linkage to disease. This is particularly challenging in areas where dengue virus (DENV) is endemic or Japanese encephalitis virus (JEV) or yellow fever virus (YFV) vaccination is common. Current recommendations from the U.S. Centers for Disease Control and Prevention (CDC) are that all samples found seropositive in a Zika virus IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) should be validated by plaque reduction neutralization testing (PRNT). PRNT is expensive and labor-intensive and requires live virus. Furthermore, antibodies to conserved flavivirus domains may confound assay specificity, particularly in the acute and early convalescent phases. Here, we report a sensitive, highly multiplexed, microarray-based assay that enables discrimination of antibody responses to linear epitopes specific to ZIKV, dengue virus (DENV) 1 to 4, chikungunya virus (CHIKV), West Nile virus (WNV), yellow fever virus (YFV), Ilheus virus (ILHV), and Oropouche virus (OROV). We also report the identification of a highly specific epitope for ZIKV located within the NS2B protein. We adapted this NS2B peptide to a synthetic biotinylated peptide IgG ELISA system with the goal of establishing an inexpensive, rapid, sensitive, specific, and point-of-care method for ZIKV IgG detection.
Arboviral peptide array design.Our platform for epitope discovery is a programmable peptide microarray that can accommodate up to 3 million distinct linear peptides on a 75-mm by 26-mm slide (Roche). The array can also be divided into 12 subarrays, each containing approximately ~170,000 "12-mer peptides." The 12-mer format is based on the observation that serum antibodies bind linear peptide sequences ranging from 5 to 9 amino acids (aa) and bind most efficiently when targets are flanked by additional amino acids (13). To enable differential detection of antibodies specific for arboviral infections in geographic regions where ZIKV has become endemic (14, 15), we created a custom NCBI GenBank database for ZIKV, DENV, CHIKV, YFV, WNV, OROV, and ILHV (Table 1). For each virus selected, we downloaded all available protein sequences available before October 2016 from the NCBI protein database. We then created a database comprising overlapping 12-mer peptides that tiled the whole proteome of each of these agents with 11-aa overlap in a sliding window pattern. The selected peptide sequences were passed through a redundancy filter to yield 88,212 unique peptide sequences and 83,748 nonunique peptide sequences (present in more than one virus) for a total of 171,960 peptides. Redundant peptides were excluded prior to synthesis. The individual peptides in the library were printed in random positions on the peptide array to minimize the impact of locational bias.
Arboviral peptide array data analysis.The array data, in the form of fluorescence signal intensities (arbitrary units [AU]), were background and spatially corrected and quantile normalized using the R Package "preprocessCore" (16). Spatial correction was based on a two-dimensional local polynomial regression (LOESS) that adjusts for signal variation due to spatial effects. A cutoff threshold for peptide recognition was defined as mean + 2 times the standard deviation (SD) of the mean intensity value of all negative controls (17). The statistical comparison of peptide microarray intensities between groups was performed using the limma R package (18). Peptide intensities for all samples were loaded and normalized using the "normexp" method for cross-array normalization (19).
Identification and selection of reactive peptides.To test the utility of the array, we used 308 sera immunoreactive to ZIKV, DENV, CHIKV, YFV, tick-borne encephalitis virus (TBEV), WNV, or JEV and 21 sera from controls from the greater New York City metropolitan area with no known history of flavivirus infection or flavivirus vaccination who had been tested for exposure to Borrelia burgdorferi, the causative agent of Lyme disease. Table 2 lists sources of sera and reference methods used to define immunoreactivity. We employed dilutions of 1:50 or 1:100 based on pilot experiments wherein intensity analysis demonstrated that 1:50, 1:100, and 1:200 dilutions with known positive sera produced signal above the threshold (mean + 2 SDs above the mean obtained with negative-control samples), whereas dilutions of >1:200 produced signals similar to those found with negative-control samples (mean + 2 SDs).
Signal data points were next filtered such that only peptides that showed signal in at least one sample (1/12 subarrays) with an intensity greater than the previously defined threshold (mean + 2 SDs, intensity of ~9,000 AU) were retained. This step reduced the initial number of peptides from 171,891 to 84,018 for data analysis. Fold changes and standard deviations were estimated by fitting a linear model for signal intensities generated by each peptide, applying empirical Bayes smoothing to the standard deviations, and then determining those peptides that yielded statistically significant signal by contrasting linear models for each peptide between conditions (20). The false-discovery rate was controlled at the 0.05 level using the Benjamini-Hochberg procedure (21). Each analysis was performed twice: once to differentiate peptides that were immunoreactive with ZIKV early-convalescent-phase versus control sera and once to differentiate peptides that were immunoreactive with ZIKV early-convalescent-phase versus DENV convalescent-phase sera. Six hundred twenty-four peptides yielded statistically significant differences (adjusted P < 0.05) in signal intensity between ZIKV convalescent-phase and control sera. Two thousand one hundred fifty-six peptides yielded statistically significant differences (adjusted P < 0.05) in signal intensity between ZIKV convalescent-phase and DENV convalescent-phase sera. Multidimensional scaling (MDS) demonstrated the capacity of these peptides to separate ZIKV from DENV sera and control group sera (Fig. 1).
Identification of an immunoreactive, sensitive, and specific peptide sequence in the ZIKV NS2B protein.For each agent-positive sample examined, we cataloged all immunoreactive IgG linear epitopes. Immunoreactivity was highly reproducible, with minimal interchip variation (see Fig. S1 in the supplemental material). Peptides were considered agent specific if they were reactive only with their cognate sera, signal intensities were 2 SDs above the threshold established with negative-control sera, and a minimum of 3 continuous overlapping peptides were reactive. Based on these criteria, 9 overlapping peptides comprising a total of 20 aa were identified as agent specific in NS2B of ZIKV (Fig. 2). Nine peptides comprising a 20-aa region in NS2B of ZIKV were observed to be highly significant in differential analysis, displayed high signal intensities, and formed an epitope of 9 continuous overlapping peptides. To examine the potential predictive power of these 9 peptides when differentiating ZIKV convalescent-phase samples (early and late) from DENV (early and late) convalescent-phase samples, classification models were created using random forest (22) with signal intensities as predictors. Signal intensity data were obtained with 108 sera previously defined as containing antibodies to ZIKV (early and late convalescent phase) by ZIKV nonstructural protein 1 blockade-of-binding (BOB-ELISA) (23) and 114 DENV (early- and late-convalescent-phase) samples. The nine peptides from the 20-aa epitope in NS2B were first ranked with respect to their measurements of mean decrease in accuracy. The nested classifiers using the top-ranked peptides were evaluated by random resampling cross-validation with 1,000 iterations. Data were randomly split into a training set (80%) and a test set (20%) within each iteration. The area under the curve (AUC) values, together with their 95% confidence intervals (CIs), were calculated, and the corresponding receiver operating characteristic (ROC) curves were plotted. The best-performing classifier consisted of the top 6/9 ranked peptides and yielded a cross-validated AUC of 0.931 (95% CI, 0.818 to 0.975) (Fig. 3).
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
Testing and validation of 20-aa ZIKV NS2B peptide.A total of 308 serum samples were tested with the arboviral peptide array: 138 for ZIKV, 124 for DENV, 24 for CHIKV, 5 for WNV, 10 for YFV, 3 for TBEV, and 4 for JEV and 21 healthy controls with no known history of flavivirus or alphavirus infection (Table 2). The ZIKV sera included samples collected from 15 patients at three time points: 1 to 6 days after onset of disease (acute phase), 2 to 3 weeks after onset of disease (early convalescence), and ~6 months after onset of disease (late convalescence). Other ZIKV sera included samples collected from individual subjects at a single time point: 15 acute-phase, 75 early-convalescent-phase, and 3 late-convalescent-phase sera. Based on previous exposure to DENV, we also divided ZIKV-positive samples into two groups, DENV immune and DENV naive. Thus, we included 30 ZIKV acute-phase (9 DENV-immune and 21 DENV-naive), 90 ZIKV early-convalescent-phase (25 DENV-immune and 65 DENV-naive), and 18 ZIKV late-convalescent-phase (8 DENV-immune and 10 DENV-naive) samples. Eighty-eight of 90 early-convalescent-phase ZIKV sera (98%) were strongly positive for the NS2B peptide with signal intensities exceeding 60,000 AU. Fourteen of 30 (47%) acute-phase ZIKV sera, 12/18 (67%) late-convalescent-phase ZIKV sera, and 1/3 (33%) JEV vaccinee sera were also strongly reactive. Samples with prior exposure to DENV had higher ZIKV reactivity with the 20-aa NS2B peptide: DENV-immune ZIKV-positive acute-phase sera had higher reactivity than DENV-naive ZIKV-positive acute-phase sera (55% versus 43%), and DENV-immune ZIKV-positive late-convalescent-phase sera had higher reactivity than DENV-naive ZIKV-positive late-convalescent-phase sera (75% versus 60%). Four of 124 (3%) DENV, 1/24 (4.1%) CHIKV, and 1/10 (10%) YFV sera were moderately reactive, with signal intensities below 20,000 AU. No cross-reactivity was observed with 12 DENV acute-phase sera, 1 JEV convalescent-phase serum, 5 WNV convalescent-phase sera, or 3 TBEV vaccinee sera. One positive acute-phase CHIKV sample from Nicaragua which was found moderately positive for ZIKV NS2B peptide was collected in November 2016 just before the first clinical ZIKV case was reported in Nicaragua (24). Detailed data for immunoreactivity, sensitivity, and specificity are given in Table 2. One of the 21 control sera from a patient residing in the greater New York City metropolitan area with an unknown travel history was also moderately reactive. ZIKV early-convalescent-phase samples showed signal intensity up to 60,000 AU for 20-aa NS2B peptides and were significantly higher (P < 0.005) than control groups except for one JEV vaccinee sample (Fig. 4A). ZIKV acute-phase and ZIKV late-convalescent-phase samples showed comparatively lower signal intensity (up to 40,000 AU) than ZIKV early-convalescent-phase samples (Fig. 4B).
African ZIKV isolates differ from ZIKV isolates from Asia and the Americas in the presence of a valine rather than an isoleucine in the 20-aa ZIKV NS2B peptide region employed in our assays (10th amino acid in 20-aa ZIKV NS2B peptide, V>I). Both peptides were printed, allowing us to determine the impact of this mutation on immunoreactivity and assay performance. We found no differences in signals in sera from subjects infected in Asia or the Americas (Fig. 5). We have no African sera for reciprocal analyses.
Development of a ZIKV NS2B peptide ELISA.Due to costs for fabrication, instrument purchase, and protocol complexity, we view peptide arrays as platforms for discovery rather than as tools for routine serology. Accordingly, we used data acquired in array analyses to build a ZIKV NS2B peptide ELISA. Twenty-four-residue peptides were synthesized that included the immunoreactive 20-aa ZIKV NS2B peptide as well as two flanking amino acid residues (underlined) at both the amino and carboxyl termini to serve as spacer molecules for epitope presentation (AGDITWEKDAEVTGNSPRLDVALD). We also synthesized peptides ranging from 8 to 14 aa within the core of the region defined as most strongly immunoreactive in Fig. 2 and a concatemer of the 24-aa peptide joined by a central glutamic acid residue (AGDITWEKDAEVTGNSPRLDVALDEAGDITWEKDAEVTGNSPRLDVALD). All peptides were synthesized with biotin on either the amino or carboxyl terminus to facilitate binding to plates coated with antibodies to biotin. The performance of the carboxyl terminus biotinylated 49-aa peptide concatemer ELISA was superior to the other peptide ELISAs. It identified 96% of ZIKV sera positive in the peptide array with 94% specificity. The 8- to 14-aa peptide ELISA had less than 50% sensitivity and 80% specificity. The 24-aa peptide ELISA had ~80% sensitivity and specificity. The amino terminus biotinylated 49-aa peptide concatemer ELISA yielded 70% sensitivity and 60% specificity. Accordingly, we are here reporting only findings with the carboxyl terminus biotinylated 49-aa peptide concatemer ELISA (ZIKV-NS2B-concat ELISA).
Validation of the ZIKV-NS2B-concat ELISA.All 329 serum samples (Table 2) previously tested with the arboviral peptide array were also tested with the ZIKV-NS2B-concat ELISA. Eighty-six of 90 early-convalescent-phase ZIKV sera (96%) were positive (optical density [OD] > 0.90) by ELISA. Eight of 30 (26.6%) acute-phase ZIKV sera, 8/18 (44%) late-convalescent-phase ZIKV sera, and 1/3 (33%) JEV vaccinee sera were also positive (>0.90 OD) with ELISA. Samples with prior exposure to DENV had higher ZIKV reactivity in ELISA also: DENV-immune ZIKV-positive acute-phase sera had higher reactivity than DENV-naive ZIKV-positive acute-phase sera (55% versus 33%), and DENV-immune ZIKV-positive late-convalescent-phase sera had higher reactivity than DENV-naive ZIKV-positive late-convalescent-phase sera (62% versus 50%). Two of 90 (2%) early-convalescent-phase ZIKV sera, 4/30 (13%) acute-phase ZIKV sera, 2/18 (11%) late-convalescent-phase ZIKV sera, 9/124 (7%) DENV sera, 1/24 CHIKV sera (4%), and 1/10 YFV sera (10%) were moderately reactive (0.65 to 0.90 OD). No cross-reactivity was observed with 5 WNV or 3 TBEV sera. One of 21 sera from the control group that was positive in the peptide array was also positive in the ELISA. Detailed data for immunoreactivity, sensitivity, and specificity are given in Table 2.
A total of 154 ZIKV sera (30 acute-phase, 70 early-convalescent-phase, and 54 late-convalescent-phase sera) were also tested side by side by the ZIKV-NS2B-concat ELISA, a commercial NS1 recombinant IgG ELISA (anti-ZIKV ELISA IgG assays; Euroimmun, Lübeck, Germany) (25), and a Zika nonstructural protein 1 (NS1) BOB-ELISA (23) (Fig. 6A). The sensitivity of the ZIKV-NS2B-concat ELISA was superior to the recombinant commercial NS1 IgG ELISA with acute-phase (47% versus 13%), early-convalescent-phase (96% versus 65%), and late-convalescent-phase (55% versus 26%) sera. The BOB-ELISA had similar sensitivity with early-convalescent-phase sera (94%) and superior sensitivity with late-convalescent-phase sera (94%) but detected no immunoreactivity in acute-phase sera (data not shown). The immunoreactivity of ZIKV-NS2B-concat ELISA with sera collected from 28 ZIKV patients over the time course of infection was higher in early convalescent phase than in the acute and late convalescent phases and consistent with findings obtained by using the peptide array (Fig. 4B and 6B).
Since the emergence of ZIKV in the Americas in 2015, 583,144 cases have been reported to the World Health Organization. On 6 April 2017, the United Nations Development Programme, in partnership with the International Federation of Red Cross, Red Crescent Societies and the Pan-American Health Organization, estimated the economic cost of Zika disease in Latin America and the Caribbean alone as up to $18 billion between 2015 and 2017 (26). Costs will ultimately be higher because sequelae of congenital infection require lifetime support and include more subtle neurodevelopmental disabilities than the severe abnormalities observed at birth, such as microcephaly (27, 28).
Accurate estimates of the public health burden of ZIKV and medical management will require easily accessible, sensitive, and specific assays for detection of infection, which has been challenging. The efficacy of molecular methods for detection of ZIKV in serum, plasma, and whole blood is limited to the first few weeks of infection. Serology has been confounded by cross-reactivity of antibody responses due to genetic similarity and structural homology of ZIKV with other flaviviruses, especially DENV. Across the entire genomic sequence, ZIKV shares 55.6% amino acid identity with DENV, 46.0% with YFV, 56.1% with JEV, and 57.0% with WNV (29). False-positive results in ZIKV ELISA and PRNT assays have been reported after natural infection with other flaviviruses as well as after YFV and JEV vaccination (30).
The advent of peptide microarrays provides new opportunities to finely map discrete regions within the proteomes of viruses and other microbes that can facilitate differential diagnosis of infectious diseases. We used highly multiplexed, programmable peptide arrays to identify discriminant epitopes for serodiagnosis of infection. The ZIKV-NS2B-concat ELISA, generated based on results obtained with the peptide array, has utility for diagnosis of ZIKV infection. In our protocol using a threshold of an 0.90 OD, the ELISA detected IgG antibodies in 47% of sera from subjects with acute disease (1 to 6 days after onset), more than 95% of sera from subjects in early convalescence (2 to 3 weeks after onset), and 55% of sera from subjects in late convalescence (more than 6 months after onset).
The specificity of the ZIKV-NS2B-concat ELISA was 94%. One of three JEV vaccinee sera was seropositive; however, a subject with natural JEV infection was not seropositive. One individual from Nicaragua with acute CHIKV infection was also seropositive. Chikungunya virus is an alphavirus that would not be anticipated to elicit antibodies cross-reactive with flaviviruses, and the samples analyzed were all collected before the introduction of ZIKV. We do not know whether this individual also had a history of exposure to ZIKV. The closest homologue in NCBI-BlastP to the 20-aa ZIKV NS2B peptide sequence is a sequence in Spondweni virus (SPONV) (17/20 aa, 85% identity) (see Fig. S2 in the supplemental material). Spondweni virus has been reported in sub-Saharan Africa; only 6 cases are documented (31). The SPONV serogroup includes both ZIKV and SPONV. We have not tested sera from individuals infected with SPONV and acknowledge that differential diagnosis may not be feasible with the ZIKV-NS2B-concat ELISA alone. JEV and St. Louis encephalitis virus (SLEV) have 80% amino acid homology (16/20 amino acid residues) with the ZIKV NS2B peptide sequence. This may account for false-positive results with sera from the subject recently vaccinated for JEV wherein the antibody titers for the 20-aa ZIKV NS2B peptide sequence were high and underscores the importance of obtaining an accurate vaccine history. We did not have access to sera from subjects exposed to SLEV to test for immunoreactivity to the ZIKV NS2B peptide. We observed lower-titer cross-reactivity that did not cross the 0.90-OD threshold for definitive serological ZIKV diagnosis with other flaviviruses, including DENV1, DENV2, DENV3, DENV4, WNV, and TBEV.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
Preexisting dengue virus antibodies may modulate immune responses to ZIKV infection (32). Priyamvada et al. have proposed that this is due to reactivation of preexisting memory B cells that target conserved epitopes (33). We found that prior exposure to DENV was associated with higher ZIKV reactivity to NS2B peptide in both the peptide array and ZIKV-NS2B-concat ELISA. We cannot ascertain whether the converse is true as we have no sera from subjects infected with ZIKV who were subsequently infected with DENV. However, we found no reactivity to the DENV NS2B using DENV sera.
One of 21 control sera obtained from patients in the greater New York City metropolitan area tested for Borrelia burgdorferi, the causative agent of Lyme disease, also had reactivity in the ZIKV NS2B peptide ELISA and peptide array. We do not have travel history or vaccine records for these control patients and cannot discern whether reactivity was due to flavivirus infection or vaccination.
The peptide array provided a modest improvement over the ELISA in sensitivity with acute-phase (47% versus 40%), early-convalescent-phase (98% versus 96%), and late-convalescent-phase (67% versus 55%) sera. It was also more specific and allowed us to resolve the JEV and CHIKV immunoreactivity observed in ELISA. Whereas all early-convalescent-phase ZIKV sera detected 5 or more peptides, each with signal in excess of 40,000 AU, the JEV vaccine and CHIKV sera detected only 3 peptides; none yielded signal greater than 30,000 AU. Nine of 124 DENV sera had intermediate reactivity in ELISA (OD of 0.60 to 0.90). None reacted with 5 peptides or had signal in excess of 40,000 AU. The single serum from the 21-subject control group that was positive in ELISA remained positive on the array (detecting 5 peptides with more than 40,000 AU). An algorithm for serodiagnosis of ZIKV exposure based on the limited sample set employed in this work is shown in Fig. 7.
ZIKV infection in any trimester can result in stillbirth or congenital birth defects (12). This includes clinically inapparent infections. It is important, therefore, that the diagnostic armamentarium for risk assessment have the capacity to detect evidence of current as well as past infection over the entire course of gestation. The ZIKV-NS2B-concat ELISA is most sensitive for detection of infection during early convalescence. We do not have the samples required to assess performance in the interval between early and late convalescence (3 weeks to 6 months); nonetheless, based on data obtained during early convalescence, monthly testing during each of the three trimesters should detect more than 95% of ZIKV infections. Where results are equivocal, the peptide array can be used to increase the sensitivity for detection of ZIKV infection to 98% and delineate triggers for cross-reactivity, including other flavivirus infections or vaccination. The ZIKV-NS2B-concat ELISA can be readily implemented with equipment employed in routine clinical microbiology laboratories found in the developing world where the burden of infection is highest. As the peptide array requires more elaborate imaging equipment and bioinformatics support for analysis, it will be important to explore whether less resource-intensive serological tests can be established that quantitate responses to the multiple peptides that enhance sensitivity and specificity in the peptide array.
The positive and negative predictive values of a serological assay vary with the prevalence of infection in the population. Our data concerning sensitivity and specificity reflect findings in subjects considered to be at high risk for infection with ZIKV or other arboviruses. Additional studies will be required to establish the positive and negative predictive values of the assays in other populations.
The peptide array platform that we employed displays only linear amino acid sequences. It is possible that a system that displays conformational epitopes might have revealed epitopes with better diagnostic performance profiles than ZIKV NS2B. We nonetheless predict that due to flexibility and high-throughput screening capacity, the programmable peptide array platform will become a vital tool in the discovery and development of serodiagnostics.
Serum or plasma samples for the study.One hundred thirty-eight serum samples were collected from 90 children in the Nicaraguan Pediatric Dengue Cohort Study (PDCS) (23) who were reverse transcription-PCR (RT-PCR) positive for ZIKV in serum and had signs and symptoms of ZIKV infection between January and 20 September 2016. Acute-phase (1 to 6 days after onset of symptoms), early-convalescent-phase (14 to 21 days after onset of symptoms), and late-convalescent-phase (~6 months after onset of symptoms) blood samples were drawn for DENV, CHIKV, and ZIKV diagnostic testing and annual sample collection as part of the cohort study (Table 2) (34). We included confirmed ZIKV cases with known prior DENV infection history status (DENV naive or DENV immune) (35). Confirmed ZIKV-positive cases were classified as DENV naive if they entered the cohort study with no detectable anti-DENV antibodies (as measured by DENV inhibition ELISA) and had no documented DENV infections (symptomatic or inapparent) during their time in the cohort. Confirmed ZIKV-positive cases were classified as DENV immune if they either entered the cohort study with detectable anti-DENV inhibition ELISA antibodies or entered the cohort study with no detectable anti-DENV antibodies and had one or more documented DENV infections during their time in the cohort study. All suspected ZIKV cases were confirmed by real-time RT-PCR in serum, using the CDC Trioplex assay, the ZCD triplex assay (36), or in some cases the CDC ZIKV monoplex assay (37), in parallel with a DENV-CHIKV multiplex assay (38). We also included as a control group a total of 124 samples with DENV (12 acute-, 29 early-convalescent-, and 83 late-convalescent-phase) infection confirmed by RT-PCR between 2005 and 2013, from the New York State Department of Health Wadsworth Center Laboratory (WC), New York City Department of Health and Mental Hygiene Public Health Laboratory (PHL) (10), Nicaraguan PDCS (34), or the Nicaraguan Hospital-Based Dengue Study at the National Pediatric Reference Hospital in Managua (39). Twelve additional DENV early-convalescent-phase sera (3 for each of DENV1 to -4) that tested positive with PRNT were collected from Thailand (40). Six acute- and 12 early-convalescent-phase CHIKV sera were collected from real-time RT-PCR-confirmed chikungunya cases in the Nicaraguan hospital-based study (41); 6 acute-phase CHIKV sera were provided by WC and PHL (10). Convalescent-phase CHIKV sera were tested for anti-CHIKV antibodies with Euroimmun anti-chikungunya virus immunofluorescent assay (IFA) (IgG). Five banked WNV early-convalescent-phase sera after natural infection were also used (10). Ten YFV (vaccinated), 3 TBEV (vaccinated), and 4 JEV (1 natural infection convalescent-phase and 3 vaccinated) sera were collected by the Erasmus University Medical Centre (Rotterdam, Netherlands). Sera from 21 subjects in the greater New York City metropolitan area tested for Lyme disease who had no known history of flavivirus or alphavirus infection or vaccination were provided by the Lyme Disease Laboratory (School of Medicine, Stony Brook University) for use as controls.
Arboviral peptide array synthesis, sample binding, and processing.Peptide synthesis was accomplished by light-directed array synthesis in a Roche maskless array synthesizer (MAS) using an amino functionalized substrate coupled with 6-aminohexanoic acid as a spacer and amino acid derivatives carrying a photosensitive 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) group. Coupling of amino acids was done using preactivated amino acid with activator (1-hydroxybenzotriazole [HOBT]/2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium [HBTU]) and ethyldiisopropylamine in dimethylformamide (DMF) for 5 to 7 min before flushing the substrate. Cycles of coupling were repeated until 12-mer peptides were synthesized. Intermediate washes on the arrays were done with N-methyl-2-pyrrolidone (NMP), and site-specific cleavage of the NPPOC group was accomplished by irradiation of an image created by a digital micromirror device (Texas Instruments; SXGA+ graphics format), projecting light with a 365-nm wavelength. Final deprotection to cleave off the side chain protecting groups of the amino acids was done with trifluoroacetic acid (TFA)-water-triisopropylsilane for 30 min.
Sera were diluted (1:50 or 1:100) with binding buffer (0.1 M Tris-Cl, 1% alkali-soluble casein, 0.05% Tween 20, and water). The peptide arrays were incubated overnight at 4°C on a flat surface with individual sample/subarray. Overnight sample incubation was followed by three 10-min washes with 1× Tris-buffered saline with Tween 20 (TBST) (0.05% Tween 20) on a Little Dipper automatic washer (catalog no. 1080-40-1; SciGene) at room temperature (RT). Secondary antibodies (catalog no. 109-605-098; Alexa Fluor 647-AffiniPure goat anti-human IgG, Fcγ fragment specific; Jackson ImmunoResearch Labs) were diluted in binding buffer at a concentration of 0.1 µg/ml, and arrays were incubated in a plastic Coplin jar (catalog no. S90130; Fisher Scientific) for 3 h at RT with gentle shaking on a rocker shaker. Secondary antibody incubation was also followed by three 10-min washes on a Little Dipper washer with 1× TBST at RT. After a final wash, the arrays were dried and read using a Roche MS 200 microarray scanner (Roche), and signals were extracted using Roche internally developed image extraction software. The array slides were scanned at 2-μm resolution, with an excitation wavelength of 635 nm. The images were analyzed using the PepArray analysis program. The fluorescent signals were converted into arbitrary unit (AU) intensity plots ranging in minimum to maximum intensity from 0 to 65,000 AU.
ZIKV-NS2B-concat ELISA.A 49-aa-long concatemer of the 24-aa peptide ZIKV NS2B peptide joined by a central glutamic acid residue and carboxyl-terminal biotin labeled with a junction lysine residue, AGDITWEKDAEVTGNSPRLDVALDEAGDITWEKDAEVTGNSPRLDVALD(Lys[biotin]), was synthesized with high-performance liquid chromatography (HPLC) purity of ≥75.0% (GenScript, Piscataway, NJ). The peptide was dissolved in 3% ammonia water and stored at −20°C in small aliquots. Peptide ELISAs were performed on Corning 96-well clear-bottom plates (Sigma-Aldrich). Preadsorbed rabbit polyclonal antibiotin antibody-IgG heavy plus light chains (H+L) (catalog no. ab53494; Abcam) were diluted with 1× ELISA coating buffer (catalog no. BUF030A; Bio-Rad), applied as a coating on 96-well ELISA plates (0.6 μg/100 μl per well), and incubated at 37°C overnight under a sealed condition. After incubation, the plate was washed three times with phosphate-buffered saline (PBS)–0.05% Tween 20 (PBS-T). Following three washes with PBS-T, plates were incubated for 1 h at RT with 200 µl/well of blocking buffer (catalog no. 37539; Thermo Fisher Scientific). Afterward, plates were washed again three times with PBS-T. ZIKV NS2B peptide was diluted in blocking buffer (0.4 μg/100 μl), and 100 μl of diluted ZIKV NS2B peptide was applied as a coating on plate-wells and incubated at 37°C for 90 min. Following 3 washes with PBS-T, serum/plasma samples were diluted 1:50 or 1:100 in blocking buffer, coated (200 µl/well), and incubated at 37°C for 90 min. After 3 washes with PBS-T, preadsorbed goat anti-human IgG H+L–horseradish peroxidase (catalog no. ab97175; Abcam) was diluted (1:5,000) and coated (100 µl/well) for 90 min at 37°C. Plates were washed again 3 times with PBS-T, 1-Step Ultra tetramethylbenzidine (TMB)-ELISA substrate solution (catalog no. 34028; Thermo Fisher Scientific) was added, and plates were incubated at RT and in the dark. The reaction was stopped with 50 μl Wash and Stop solution for ELISA without sulfuric acid (catalog no. MK021; TaKaRa Clontech). Plates were read on an automated plate reader at 450 nm. The OD readings represent the reactivity of peptide ELISA. The ELISA cutoff was established by running 48 healthy control human sera with ZIKV-NS2B-concat ELISA. The nonparametric method of two times the mean of ODs (0.65) of the sera from uninfected healthy controls was established as moderate positive, and three times the mean of ODs (0.90) of the sera from uninfected healthy controls was used as the definitive positive cutoff (42).
We are grateful to the patients and clinicians who provided samples without which this work could not have proceeded. We are also grateful for technical support from Dominick Centurioni and Amy Dean (New York State Department of Health) and Dakai Liu, Taryn Burke, and Aaron Olsen (New York City Department of Health and Mental Hygiene). We thank past and present members of the study team based at the Hospital Infantil Manuel de Jesús Rivera, the National Virology Laboratory in the Centro Nacional de Diagnóstico y Referencia of the Nicaraguan Ministry of Health, and the Sustainable Sciences Institute in Managua, Nicaragua, for their dedication and high-quality work.
We are grateful for financial support from National Institutes of Health award U19AI109761 (CETR [Center for Research in Diagnostics and Discovery]). E.H. received funding support from National Institutes of Health grants P01AI106695, U19AI118610, and R01AI099631 (to A.B.). C.B.E.M.R. received funding support from ZIKAlliance H2020 (73548). N.M. was partially supported by 2016–2017 Calderone Junior Faculty Awards awarded by the Mailman School of Public Health, Columbia University.
The manuscript has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.
Eric H. Sullivan and Jigar J. Patel are full-time employees of Roche, Madison, WI. The other authors declare no competing financial interests.
- Received 26 January 2018
- Accepted 30 January 2018
- Published 6 March 2018
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.


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McCourts Gearing Up for Divorce Battle
Jamie McCourt has no interest in surrendering her ownership involvement with the Dodgers and has no plans to leave her post as chief executive officer, her attorney said Thursday.
The statements set the stage for what could be a protracted battle for control of the club between Jamie McCourt and her husband, Frank, who announced their separation late Wednesday.
Although Frank McCourt is listed as the owner and franchise "control person," to major league baseball, the source said the couple presented themselves together for the approval of Commissioner Bud Selig, in advance of the McCourts' 2004 purchase of the Dodgers.
Marshall Grossman, the attorney for Frank McCourt, contend his client is the sole owner of the Dodgers. "We disagree with his conclusion," said Dennis Wasser, the attorney for Jamie McCourt. "We are confident that, if the ownership issue must be adjudicated, the Dodgers will be determined to be community property, owned 50 percent by each of the McCourts."
- Read the full Los Angeles Times story.
For reprint and licensing requests for this article, CLICK HERE.
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- Dodgers Sale to Magic Johnson Group Completed
- Dodgers Ordered to Release Details of McCourt Deal
- The 50 Wealthiest Angelenos: Frank McCourt - #40
- The McCourts: 'Til Debt Do Us Part

subset: commoncrawl


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- Home
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Pre-heat your oven to 180 degrees Celsius.
Combine the squid, potatoes, onions, garlic, chilli, JOSEPH olive oil and oregano in a large bowl and mix well.
Tip contents of large bowl into a shallow (5cm) baking dish and cover with foil. Place baking dish in the oven for one hour at 180 degrees Celsius.
Increase the oven to 220 degrees Celsius, remove foil from the baking dish and continue cooking until the mixture is browned on top - this should take another 20 minutes or so.
Remove baking tray from oven and season the dish with sea salt and freshly cracked black pepper.
Sprinkle parsley and lemon juice over the top and stir the mixture thoroughly so that the potato starts to soften and combine with the JOSEPH olive oil and lemon juice.
Serve with a simple green salad, crusty bread and a bottle of Primo & Co 'The Venetian' Garganega!
Buon Appetito!


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For additional information, read the Nomination Form PDF
VLR Listing Date 06/12/2002
NRHP Listing Date 09/14/2002
NRHP Reference Number 2000978
Roanoke Downtown Historic District is located in the southwest quadrant of downtown Roanoke. The city became the principal center of commerce, transportation, and industry following the location of the Norfolk & Western Railway headquarters there in the early 1880s. Situated west of the Roanoke City Market Historic District, north of the residential Southwest Historic District, and south of the Roanoke Warehouse Historic District, the Roanoke Downtown Historic District contains the financial, commercial, and governmental center of the city. Seventy percent of the district's 144 primary buildings are identified as contributing to the late 19th- to mid-20th-century architectural character of the district.
In 2007, the boundary of the district was expanded to encompass four commercial buildings on Salem Avenue constructed between the years 1906–1938. The buildings are typical in style and detail of small-scale commercial and industrial buildings in the downtown area in the early 20th century. The historic uses of these buildings—which include an electric motor shop, a sheet metal shop, a candy factory, and an upholstery repair shop—relate to the commercial and retail business of the downtown area, as well as the shipping and industrial activities of the railroad and its related industries. These buildings help to illustrate the commercial, shipping and industrial development and architecture of Roanoke from the late 19th century to the mid-20th century.
128-5761 Roanoke Downtown HD 2007 Boundary Increase
In 2013, update and adjustment further carried forward the district's period of significance to 1961. That year saw the construction of the Thomas B. Mason Building, and marked the last major building constructed in downtown Roanoke prior to the City's economic downturn and the decentralization of commerce to suburban shopping centers in the early 1960s. The nomination update expanded the areas of significance to include the areas of Religion and Recreation with the addition of several downtown churches and the YMCA Building in the expanded boundary area.
128-5761 Downtown Roanoke HD 2013 Boundary Increase and Update
VLR: Virginia Landmarks Register
NPS: National Park Service
NRHP: National Register of Historic Places
NHL: National Historic Landmark
Updated August 7, 2018


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Verizon FiOS Gives Funimation Channel Back!
Do you recall when Verizon ruined the fun of its subscribers with the dreadful news that it is cancelling the Funimation Channel and delete it from its list? Well, today the news of its return was announced! Yes, Funimation Channel is coming back to its fans from North America.
FUNimation Channel HD will be available to everyone of the 4,000,000 subscribers of Verizon FiOS through their on-demand service. The current anime provided by Funimation Ch. include Fullmetal Alchemist: The Sacred Star of MIlos, Vandread and others.
Source: Anime News Network
Is this great news or not-so-great? What do you think? Comment below.


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Stripped down to just voices, guitar and vintage drums, Hat Fitz & Cara embellish impeccable rootsy originals with powerful and soulful vocals reminiscent of a time once forgotten. They have been referred to as festival favourites globally, as they share a personal invitation into the parody of their lives on the road together that often has their audiences in tears of laughter.
Hat Fitz & Cara have been known to pack more energy, emotion and sheer musical exuberance into one song than most manage in a whole career. Their music comes straight from the heart with a sound that is full of inherent realism, making their live shows visually electric.
Hat Fitz & Cara have released their 4th album. Their last album, "Do Tell", was no1 for 3 months in the Australian Blues & Roots charts and entered the Amazon charts at No 17 in Europe. Do Tell was voted best female vocal in 2015 Chain awards Australia. They represented the South East Queensland Blues association in the semi finals of the 2015 International Memphis Blues challenge.
Tickets – $20 general/$15 CBS members
Contact 0418 633 239 or 0419 607 710


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Product Description
Selemix 7-414 Epoxy Vinyl Primer is 2 pack primer that can be applied in a wet on wet paint system with a minimum of 2 hours between coats.
Please note, the Hardener is not included with this product. Compatible Hardeners are Selemix Direct 9-010 Hardener or Selemix 9-011 Hardener.
Selemix 7-414 Epoxy Vinyl Primer is 2 pack primer that can be applied in a wet on wet paint system with a minimum of 2 hours between coats saving valuable time on application reducing the overall labour costs. Additionally it can be left for up to 2 months between coats without the need for sanding when recoating meaning items can be primed stored and coated at a later date.
When used a part of a Selemix paint system with the correct surface preparation and top coat the Selemix 7-414 is ideal for application on to bare steel, black steel, phosphate steel, galvanised steel, stainless steel, phosphate steel, fibre glass reinforced polyester, agricultural equipment, tractors, trailers, harvesters, ploughs, machine tools, milling machines, lathes, drill presses, workshop equipment, cars, trucks, steel structures, garden furniture, bicycle frames, motorcycles, plant equipment, excavators, screed pumps, power barrows, steel doors, garage doors plus many more.
Best Uses
Selemix 7-414 Epoxy Vinyl Primer is best used as a primer coat in a Selemix paint system to provide protection from the elements. Using as part of this system makes the product perfect for application on to bare steel, phosphated steel, stainless steel, galvanised steel, steel structures, machine tools, drill presses, lathes, milling machines, brake presses, guillotines, plant equipment, screed pumps, diggers, dumpers, excavators, earthmovers, power barrow, steel doors, garage doors, roller shutter doors, garden furniture, welfare cabins, electrical cabinets, airport ground support equipment plus many more.
The substrate will need to be suitable abraded and degreased in preparation of receiving a painted finish Selemix 7-414 can be applied by conventional spray equipment. Selemix 7-414 has a theoretical coverage of 5-5.2m2 per litre when applied at a 50 micron dry film thickness.
Selemix 7-414 Epoxy Vinyl Primer is a 2 pack paint meaning it will need to combined with Selemix Hardeners 9-010 or 9-011 at a general ratio of 4 to 1.
Selemix 7-414 Epoxy Vinyl Primer will also need to be thinned with Selemix Thinners 1-410 or 1-400 prior to use, always consult the technical datasheet prior to use for applicable ratio.
Technical Details
Compatible Thinners
Selemix 7-414 Epoxy Vinyl Primer will need to be thinned with either of the below prior to use:
Compatible Hardeners
Selemix 7-414 Epoxy Vinyl Primer is a 2 pack paint meaning it will need to combined with one of the below hardeners at a general ratio of 4 to 1:
- Selemix 7-414 is a 2 pack epoxy vinyl primer
- Solvent based
- High build
- Pot life 8-10 hours at 20 degrees Celsius
- Can be applied wet on wet
- Recoatable without sanding after minimum 2 hours and maximum 2 months at 20 degrees Celsius
- Touch dry 2-3 hours at 20 degrees Celsius
- Ideal for agricultural and plant equipment
- Theoretical coverage of 5 – 5.2 m2 per litre when applied at 50 micron dry film thickness
- Available in 5 and 20 litres.
Data Sheets
The manufacturers data sheets are available for download from this website. It is your responsibility to read and understand all aspects of the data sheets before you place an order and prior to application. Please pay special attention to the preparation, mixing and application temperatures of the product, cleaning of substrate and environmental factors such as temperature, rain etc. Quoted coverage rates are based on the manufacturers theoretical rates and may vary depending upon substrates absorption, texture and application. We always recommend a test is carried out to ensure suitability.
Customer further agrees that Promain have no liability for any errors or omissions contained within the Product Data Sheets and are not liable for failure due to misapplication or inadequate substrate cleaning and preparation. For more information please refer to our Terms & Conditions.


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To be the most trusted and informed recruitment advisors – with our values of honesty, transparency, respect, humility, and self-improvement. These guide us through every step of our service to candidates and clients. The result we aim for is a rewarding experience that provides genuine value.
Our investors are Recruitment Entrepreneur, one of the fastest-growing recruitment investment firms in Europe. Founded by James Caan CBE in 2014, it supports a growing portfolio of 17 partner businesses across multiple global locations.
We care about our values and believe in doing what we say we'll do. That's why we live and breathe ours in everything we do.
That means choosing honesty, ensuring all stakeholders know where they stand. And when we talk about the importance of teamwork, we mean it, that's how we've fostered a genuinely collaborative environment. We encourage humility, respect and self-improvement as these values keep us grounded.
We respect all our values and apply them relentlessly. So, we make sure we're held accountable to every single one.
Like some of your peers, you may be looking to change jobs or return to consulting. Maybe it's the diversity of projects or the opportunity to spend less time away from home.
You may be in it for the training, the structured progression, or the chance to work with other brilliant people. Whatever your motivations for moving - we'll get you on your way.
Internal Audit, Risk & Compliance are critical areas concerned with the smooth running of business operations. If you work in this space, you have to be flexible and adaptable so you can meet the rapidly changing and emerging risks of international business.
Our specialist consultants have growing networks which we constantly build and nurture, so we're able to find and place the most suitable people into these roles. If you're a C&I professional looking for your next move - get in touch for solid, confidential career advice.
I started my recruitment career in 2007, after almost graduating as a qualified carpenter. I joined a large FTSE 250 listed global recruitment company, before moving to Sydney to recruit accountants there. In 2012, I started placing auditors and advisory professionals worldwide.
At Hanami, I lead the Professional Services team. As a unit, we've placed Internal Auditors, External Auditors and Advisory (Risk Advisory, Corporate Finance, M&A, Accounting Advisory, etc) candidates into 90 cities in 39 countries worldwide. We are truly international, living and breathing the cultures of most of the 49 different nationalities we've placed. I learn something new every single day and love to travel to meet our clients and candidates – something I'll personally never tire of doing.
Besides securing new lives for the above placements which is in itself, a genuinely awesome feeling, my daily mission is for us all to laugh. A lot.
Audit and Advisory professionals worldwide
Daintree Rainforest in Queensland (never placed there sadly!) - favourite 'placed' destination is Bermuda
Take a gap year with the family and travel the world in a camper van
My children Evie and Olly. Liverpool FC. No time for anything else!
I graduated many moons ago from Sheffield University with a Degree in Modern History, with my dissertation being on "The History of the English Public House (Pub)". After teaching around the world, including four years in Japan, I moved into the world of recruitment in a market that combined my international and people-focused background.
Since 2006, I have recruited globally into all corners of the world, from Mumbai to Sao Paulo. My passion for recruiting internationally led me to co-found Hanami in 2014 and has helped in the growth of the business over the last five years.
When not recruiting, I spend time with my young family and in B&Q. When not in the local hardware shop, I like to spend time socialising, travelling and trying to keep fit.
International Audit, Controls and Investigations
Travelling through India
Music, travel and learning
Senior Consultant
I started my recruitment career in 2015 after graduating from the University of Nottingham with a 2:1 in Social Policy. I joined a large, FTSE 250 listed global recruitment company, before joining Hanami International 18 months later.
At Hanami, I've recruited Auditors and Forensic Accountants into 14 cities in 8 countries across North America and Western Europe. Some of my recruitment highlights whilst working here are helping an American forensic consultancy enter the Swiss market with a key senior hire, and relocating 13 people to the Netherlands with my Big 4 client in Amsterdam.
I frequently travel to Switzerland, Netherlands, France, and other Western European countries, so if you're looking to secure an international move in consulting, drop me a message.
Audit and Forensic Accounting
New York (although Bermuda is a close second!)
To swim with whale sharks in the Gulf of Mexico
Playing guitar, scuba diving, and watching Arsenal FC
Senior Consultant
I started my recruitment career in 2005, after studying Geography at the University of Edinburgh. I joined an international finance recruitment company, which is where my love for cross-border recruiting began. Since then, I have helped 200+ finance professionals to move around the globe!
I have travelled to some fantastic places in my job, from Luxembourg to South Africa and Paris to The Bahamas. Meeting candidates who have relocated and are living their best lives is the most rewarding part of what I do.
At Hanami, I specialise in Financial Advisory appointments at all levels from Newly Qualified up to Partner. I work with clients ranging from small boutiques to large international accountancy practices around the world.
Corporate Finance & Financial Advisory Services
To visit all 7 New Wonders of the World
Travel (39 countries and counting), photography and cooking
Senior Consultant
I started my recruitment career in 2017 after graduating from the University of Warwick with a 2:1 in Biomedical Science. I initially joined a larger recruitment company and then came across Hanami and joined in early 2018.
Since working here I've been responsible for the growth and development of our corporate finance desk, recruiting within the Transaction Services, M&A, and Strategy teams of our clients within the professional services sector.
One of my highlights so far has been travelling to places throughout Europe that I hadn't been to before including Germany and Luxembourg. As well as this, I've also helped a client develop their teams when expanding into Europe.
You can usually find me planning my next adventure to some far-off location, so if, like me, you want an international adventure then get in touch.
Transaction Services and Corporate Finance
Noosa Heads, Australia
To go to Everest base camp (or even up it one day!)
Skiing, surfing, and most sports in between
Senior Business Support Executive
I joined Hanami in 2018 as their business support executive after spending six months in sunny Ibiza. Prior to this, I've had experience in office management in other recruitment companies.
Being the only non-recruiter in the company, I assist Andy and Matt in all aspects of the business, including HR, Operations and looking after the team.
My day to day job varies depending on the needs of the business but I love organising our monthly socials and cultural Fridays. My highlights would be Oktoberfest and visiting the Imperial War Museum.
Hanami International is a really special place to work. Like a big family, I'm surrounded by the best and most supportive recruiters and learn from them on a daily basis.
Keeping the business running
Es Vedra, Ibiza
A safari
Socialising with friends, gym (some of the time) and music
Having graduated from Loughborough University, with a 2:1 in Construction Engineering, I worked through the management training graduate scheme in positions in both the head office and in operations for 4 years.
I joined Hanami shortly after its inception in September 2014, recruiting internal audit, risk, and finance and accountancy professionals into cities all over the world. I've travelled frequently to more than 10 countries with work and placed people at all levels, from those new to the profession, to those at the very top end of the market in leadership positions of FTSE 100 and Fortune 500 businesses. I started managing other recruiters in 2017 and I'm extremely proud of the work my team does to carve a market in a growing, competitive and professional space.
Internal Audit and Risk
I love to travel, but London will always be my favourite city
To visit every country in the world
Travelling, sport, and property development
After finishing my A-Levels in Maths, Business Studies and Physical Education, I took a year out to go travelling with a key highlight being my three month trip to India teaching sport.
My recruitment career started in 2018 when I joined the internal audit team for a financial services recruitment company. I went on to join Hanami just over a year later. My role here involves helping internal auditors move into or within industry across the UK & Ireland at all levels.
If you are currently based in looking for a new internal audit position within the UK & Ireland then let's talk.
Internal Audit
Kerala, India
To climb the Rainbow Mountain in Peru
Cricket, football, rugby, running and travelling
After finishing my education in Plymouth and completing a BTEC in sports, I travelled to Australia for 4 months on a 'rugby exchange'. Shortly afterwards, I was offered a contract to play rugby back in the UK, in Plymouth. I went on to play professional rugby for two years, before deciding to move to London to be closer to family and friends.
I started working in recruitment in 2018 when I joined a leading recruitment business in the education sector, where recruited for lecturers within further education colleges all over London.
After 18 months recruiting within education I then went on to join Hanami, where I am currently responsible for recruiting Auditors within the Big 4 internationally, with no geographical limitations!
International Audit within Professional Services
To travel Asia
Rugby, socialising with friends, and trying to keep the streets of London safe as a special constable with the Met Police
I joined Hanami International after 2.5 years working within Accounting and Finance within the UK market (London & Surrey/Sussex).
My focus at Hanami is to recruit Internal Auditors into a variety of businesses within Commerce & Industry with an European remit. Having previously travelled extensively across Australia, Europe, South, Central, and North America it seemed like a natural progression for me to join such a multi-national business!
I'm always happy to speak if you are looking for a role within Internal Audit in Europe, so please don't hesitate to get in touch
International internal audit, commerce and industry
Skiing in Meribel
Road trip through Alaska
Watching Rugby, cooking new dishes, keeping fit & healthy
Smile Train is an international children's charity that supports 100%-free cleft treatment for children globally. By using the sustainable 'teach a man to fish' model, Smile Train focuses on training local medical professionals to perform cleft repair surgery and provide comprehensive cleft care in their own communities. As a result of Smile Train's efficiency, the organisation have supported more than 1.5 million cleft surgeries since 1999.
Smile Train is an international children's charity that supports 100%-free cleft treatment for children globally. By using the sustainable 'teach a man to fish' model, Smile Train focuses on training local medical professionals to perform cleft repair surgery and provide comprehensive cleft care in their own communities. As a result of Smile Train's efficiency, the organisation have supported more than 1.5 million cleft surgeries since 1999.


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Tilda with Blackie the cat | By Tiziana
Hi, Tiziana here and today I've made a new page in my planner. I used the stunning Magnolia Stamp "Tilda with Blackie the cat" From Bugs Kisses & Halloween's collection. I used my Distress Inks to colour her.
The paper chosen is from Maja design and is called Christmas Season and the monochrome paper is always by Maja design.
The dies I chose to use are from Magnolia, the stamps on the tags are old and are part of my stash.
Finally to embellish it all I put lace, flowers, half beads, paper doily and ribbon.
Links to Products I used:
Maja design Christmas season 6x6 pad
Maja design monochromes shades of christmas
Tilda Halloween wishes - tilda with blackie the cat
Magnolia stamps doohickeys
Embellishments ribbons and threads
Embellishments - flowers
I wish you a happy day, I hope you enjoy my project. You can find my personal blog here.
See you soon
Swedish House Crafts is pleased to announce the wholesaler and retailer for Paper Nest Dolls in UK and Europe. For wholesale enquiries, please visit: https://www.swedishhousecrafts.co.uk/wholesale If you have any questions please contact: firstname.lastname@example.com


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Capturing your home's personality in quality carpentry
There's a certain element of class which you just can't get from most materials. When you're looking for that real sense of atmosphere, that personality which speaks to the senses of everyone around, you just can't beat a good piece of carpentry. How many times have you stopped to appreciate a metal bookcase? We're willing to bet not often. And when a piece of furniture has been made to our high standards, you can be sure of projecting the right feel into the room around it. That's the great thing about carpentry: you can make furniture to suit any room, any home and any layout. The only universal truth is high quality.
Crafting masterpieces for your home
You might not have thought about the sheer range of places where you can use a good piece of carpentry in the home. The first thing that most of us think of are bookcases, and we can certainly do that. But what about doors, floors, kitchen units and bathroom furnishings? We do all of these and more. If it can be done with wood, we'll be able to find a way to make it fit into your home. If you've got something on your mind, give us a ring and we'll see what we can do about it.
Setting up your office
The last thing you need is to walk into work every day and feel like you're spending the day in a soulless environment without any character. Bare and gloomy workplaces don't help anyone. We manufacture tailored office furniture to make your office a more pleasant and comfortable place. Lots of us are working from home these days. If you're looking for a great working space, you should consider our log cabins. An existing home office can be enhanced with some of our high quality furnishings as well.
A great way to bring atmosphere and character to a room. Floor to ceiling, stand-alone, contemporary or traditional – our designer bookcases can be made for any situation
More than just a place to store your clothes – we'll give you a wardrobe which has a real presence and positive effect on the room around it. We'll also ensure that it's got all of the internal draws and features you need.
Whether you're working at home or in your company's offices, you don't want to spend your day in a dull and sterile environment. Specially designed office furniture will make your workplace more comfortable and attractive.
Great to look at, easy to clean and incredibly robust – wooden floors aren't just an aesthetically pleasing choice, they're practical as well. We cover both design and installation of wooden flooring.
Doors take a fair beating over time because they're in constant use – so why not have a stronger door to start with? We manufacture both doors and window frames to individual specifications.
Complete Rooms
Want to avoid the hassle of getting several different contractors? With our complete fitted kitchens and bathrooms you'll not just get a beautiful room designed especially for you, we'll provide all the trades to fit it!
Give yourself a kitchen which is a joy to work in. With designer kitchen units made to fit both the space available and the character of your home, we'll make sure you've got a kitchen to be proud of.
It may be the smallest room but that doesn't make it any less important! Our bathroom fittings will help to make your bathroom comfortable and give it a twist of individuality.
Please call our office on 01992 577 453 within office hours for non-emergency repairs and quotations.


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Sunday, June 28, 2020
Petition Seeking Review of Perjuring Priest Bob Malm's Conduct Passes 16,000 Signatures
Tuesday, June 9, 2020
Episcopal Church Launches Abuse Survey
Friday, December 20, 2019
We're Episcopal, Aren't We? Is Grace an Episcopal Church?
There's a good article on the Wartburg Watch this week, in which friend and fellow blogger Dee Parsons asks the question, why are so many SBC churches in the closet about their SBC affiliation? That got me to thinking—-what about churches that claim to be part of a denomination, but really aren't? In other words, is Grace really an Episcopal church?
Tuesday, May 14, 2019
Protect Children: Ban the Episcopal Church!
Sunday, February 17, 2019
Article on #Churchtoo
Thursday, August 30, 2018
Amusing Discovery
Turns out, the most popular search phrase this morning was "Episcopal church in the toilet." LOL
I don't know that I've used the word "toilet" anywhere on my blog, but I still got a good laugh.
Here's what came up when I clicked through the referring URL; the guest was using Bing:
Friday, June 29, 2018
Two Years Later, Workplace Culture in The Episcopal Church is Still Broken
The Episcopal Church Center has a workplace culture marked by "fear, mistrust and resentment," according to staff and directors who answered a survey in the wake of a misconduct scandal and two high-level firings.
In the survey, released Sept. 15 at the House of Bishops meeting in Detroit, employees said they face expectations to avoid confrontation, withhold input, and strive to make good impressions, rather than do what's right. Another theme: staff find it difficult to maintain personal integrity while working for the national church.
"I'm not sure I found a sadder finding, except for the score on people not feeling that they were well-respected," said the Rev. Gay Clark Jennings, president of the House of Deputies.
Consultants from Human Synergistics, a human resources firm, shared the results with bishops gathered for their fall meeting and with members of the House of Deputies, who tuned in via webcast. Presenters laid bare how the workplace culture at 815 Second Avenue in New York City is exactly opposite of the collaborative, constructive one the employees say they want.
"This is tough stuff," said Tim Kuppler, director of culture and organization development at the consultancy. "These are the things standing in the way of accomplishing what we're talking about with the Jesus Movement."
Presiding Bishop Michael Curry brought in Human Synergistics after an independent, four-month investigation found misconduct by two senior administrators, Sam McDonald and Alex Baumgarten, had gone unaddressed before Bishop Curry took office on Nov. 1, 2015. McDonald and Baumgarten were fired in April, and the nature of the misconduct has not been disclosed.
In announcing the firings, Curry said the work ahead "is not primarily organizational and structural, but deeply cultural and spiritual." The survey begins a retraining process that will include every employee of the Domestic and Foreign Missionary Society.
Survey results pointed to dynamics in which misconduct and other misbehavior could fester. Staff said, for instance, that when they have concerns, they're expected to keep those to themselves and not speak up.
For his part, Curry sought to reassure bishops and deputies that their church's staff problems do not make it an outlier.
"The Episcopal Church is no different than any other church, all right? — so don't get depressed," Curry said. "Christianity is dysfunctional. That's just the name of the game. I mean, it's called being human. How do we get from where we are to where Jesus the Christ is actually calling us to be?"
Consultants also reassured church leaders that an organization's culture can change. Staff, supervisors, and executives will be encouraged to adopt behaviors that show respect and help achieve the culture they say they want.
Resolving to do better has already begun. President Jennings said the officers — Curry, Jennings, General Convention Executive Secretary Michael Barlowe, and Treasurer Kurt Barnes — along with three canons to the presiding bishop have made a series of commitments to one another. Among the pledges: to make decisions by consensus.
"We have committed to one another and to those with whom we work to find healthy, productive, frankly non-threatening ways to deal with those times when we are in conflict or disagree with one another," Jennings said. "Disagreement is one of the ways that we can experience resurrection and new life."
Last spring, an independent audit found the Episcopal Church needs new policies and procedures in order to protect whistleblowers. In Thursday's two-hour session, none of the speakers mentioned misconduct or how a reformed culture might include new whistleblowing safeguards.


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"Toad's Girlfriend!" is the 160th episode of SML Movies.
Toad gets a girlfriend! And she has a problem.
The movie starts with Bowser Junior about to watch the latest Doofy the Dragon episode, which had Doofy's ex-wife announcing that she had broke up with Doofy because he had cheated on a game of cards, but Doofy then ends up beating up his ex-wife, which Junior enjoys. Toad arrives texting his girlfriend.
Toad talks to Bowser Junior about his girlfriend Toadette, and Junior asks Toad to invite her over. Toadette finds Junior hot and Junior makes Toad leave by asking him to go to Cody's house to get his Thomas toy.
Toadette then kisses Junior and Toad returns from his trip. Toad mistaked their kissing for a game of Tag. Junior then asks Chef Pee Pee about girl advice, and Junior tells Pee Pee about Toadette being a cheater. And so Pee Pee tells him to tell the truth because if he dosent tell Toad, their friendship would get broken.
Junior then asks Toad to talk to him in private. Before they were going to talk, Toad points out if Toadette were to ever cheat on him, that he would kill her and the man who she is dating. This scares Junior so he changed what he was going to say for a game of tag.
Junior returns to Pee Pee. Junior admits that he did not tell Toad, so Pee Pee tells Junior to not tell him ever. Bowser Junior then fears that Toadette might tell on Toad about their kissing.
It then shows Bowser Junior kissing Toadette again. Junior returns telling Pee Pee that he could not tell Toadette. Pee Pee then questions him about his friendship with Toad. Pee Pee advises Junior to tell Toad again or else he would have to.
Bowser Junior then tells Toad about Toadette and how she is cheating. Toad, in a fit of rage, said he thinks that it's all a fantasy made up by Junior. Toad does not believe Junior and Junior promises to never talk to Toadette. The next Scene shows Junior and Toadette having a phone call. Toadette then asks Junior what he is wearing and Junior responds back by saying he wears a bib.
Junior asks her what she is wearing and she responds with being naked. Junior is disgusted by this. Toad then walks in to apologize about him getting angry. Toad then asks Junior who he is talking to. Junior says he is talking to his grandma. Junior then tells Toad to stop bringing Toadette over to his house because he believes that she is a bad person to Toad and he suggests to him to break up with her.
Junior then invites Toad to play some videogames as Junior asks Toad about what Toadette is doing. Junior then brings up about Toadette's antics. Toad then wants proof about Toadette being a cheater.
So Junior tells Toad to invite her over and for him to hide behind the pillow of the couch so he could catch Toadette kissing Bowser Junior. Toadette arrives and Junior tells her that Toad is gone to do some errands and he will be away for awile. Toadette thinks that Toad is behind the pillow but Junior says that he is not and Toadette believes him. Junior then asks Toadette if she wants to kiss. Toad comes out and calls Toadette out and thanks Junior.
Toad then breaks up with her and then Toadette wants to date Bowser Junior Toad tells Junior to make his choice, Friend or Hoe. Junior then thinks about it and he finds this a tough decision. Junior is pressured and picks Toad and shoos Toadette. Bowser Junior promises to never have a girl come between their friendship. They hear a knock on the door and Joseph appears with his girlfriend. The video ends with Junior kissing Joseph's Girlfriend.
- Bowser Junior
- Toad
- Toadette
- Chef Pee Pee
- Doofy The Dragon
- Doofy's Ex-Wife
- Joseph (cameo)
- Joseph's Girlfriend (cameo)
- Bowser (On Poster)
- Cody (mentioned)
- Margaret (mentioned)
- The game Bowser Junior and Toad are waiting to update is Super Mario Maker.
- This is Doofy's ex-wife's first visual appearance in a video after being mentioned in previous videos that feature Doofy.
- This is Toadette's first major appearance in an SML Movie.
- When Junior is talking to Chef Pee Pee, you can see custom SML and YouTube signs in the background. These signs are part of Logan's fan mail post-video.
- The poster from The Perfect Plan! is seen once again.
- This video is tied with Bowser Junior's Candy Bar! for the most lessons in one SML video with 3.
- Toads Girlfriend was uploaded one day before the Paris,France terror attacks happened.
- Toadette's name highly refers to Odette, the tough female custodian with a limp from the 2017 film Leap!
- The video's original ending was supposed to end with Junior ending up sitting between Toad, Toadette, Doofy, and Doofy's ex-wife, while Chef Pee Pee is supposed to throw them out of the house, but this was cut due to time restrictions and matured behavior that is inappropriate for kids.


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September 28, 2019
All event times are Eastern Time Zone unless otherwise specified.
NAWM Board Meeting: 8:00AM - 12:00PM
Class Agent Forum: Detchon Center International Hall, 8:00AM
Detchon International Hall
Golf at DePauw Golf Classic: Deer Creek Golf Club, Greencastle, IN
Kane Society Reception: Caleb Mills House First Floor, 10:00AM - 11:00AM
Kane Society Reception
Hosted by Advancement
Soccer vs. Wooster: Fischer Field, 12:00PM
Homecoming Luncheon 2019: Allen Athletics and Recreation Center Knowling Fieldhouse - East Court, 12:15PM - 1:30PM
Homecoming Luncheon 2019
Football vs. Denison (Homecoming): Allen Athletics and Recreation Center Hollett Little Giant Stadium, 2:00PM
'shOUT Homecoming Reception: Lilly Library 1832 Brew Lounge, 5:00PM - 7:00PM
Annual 'shOUT Homecoming Reception for alumni, students, faculty, and staff
Glee Club Homecoming Concert: Fine Arts Center M114 Salter Hall, 7:30PM - 9:30PM
Celebrate Homecoming with the Glee Club and the T-Tones! This is a family-friendly event.


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Dear Church Family,
Our scripture today comes from
James 3: 2-6.
"Now if we put the bits into the horses' mouths so that they will obey us, we direct their entire body as well. Look at the ships also, though they are so great and are driven by strong winds, are still directed by a very small rudder wherever the inclination of the pilot desires. So also the tongue is a small part of the body, and yet it boasts of great things. See how great a forest is set aflame by such a small fire! And the tongue is a fire…"
There is the old saying that "actions speak louder than words" and it can be very much true. But James reminds us that, in sin, the opposite can also be true. How many of us have thought that as long as we didn't hit someone or steal from them we have not wronged them? That we should be allowed to go home and say whatever we want about them? That, in fact, our hateful talk might even be a good thing to do so that we will keep from physical action! Or how many of us have spoken to a spouse or our children with hatefulness but then passed it off that we just have a temper or that "it was only words." But remember what James says. He reminds us that where the tongue leads so the rest of the body will follow and what the tongue starts, that fire can burn everything to the ground. James reminds us, in fact, that more than any other part of us, we need to watch our tongues.
It is like what happened in the car the other day. It was a beautiful afternoon and the weather was warm. The kids were giggling and playing in the back seat when suddenly my son shouted, "Mom, stop the car!" In a panic I quickly slowed down. It turns out, he had thrown his sister's flip flop out the car window. I was furious. I yelled threats. I shouted doom upon them. All the while, in my head, a voice said, "Stop this. Stop speaking like this. It is only a plastic sandal." Even as I heard that voice, my own voice kept on spewing. Back and forth I went: "Stop this. Stop your tongue" even as I could not stop my tongue. It is the very essence of Paul's "I do not do what I want but the very thing I do not want, that is what I do". Except it was not "doing". It was speaking. But even though it was "just words" it was sin just the same.
Today, let us all be careful what fires we set with our tongues. As James writes, Where our tongues leads, the rest of us follows. More even than any other part of us, our tongue can lead us astray. Let us pray for guidance and strength today for our tongues.
Lord Jesus, help me to hold my tongue today. When I forget, remind me that my words are powerful and can lead my whole life in the wrong direction. Guide me and my speech this day. For I need you every hour. Amen.
In Christ,


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Live Result: Here (slow sikit, Flash)
More news on Malaysian Open: Here
New format for so called Super Series really excited badminton fans (at least in Malaysia), only the best player can enter the main draw and the draw is not considering the world ranking and nationality even in the 1st round. Also, there will be Super Series Ranking and Super Series Final at the end of the year, cool..! Hope the gamble by BWF will pay off to attract views around the world via ESPN or/and StarSport.
Ong Beng Hee cruised into the second round of the US$80,000 Windy City Open squash tournament. - TheStar
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An icon is a symbol to represent something. To appreciate and recognize achievement seseorang dalam sesuatu bidang kita juga akan menggelarkan beliau as an icon. When I heard anybody mention squash, definitely I will imagine Nicol David holding the giant world cup trophy. So, I believe she is the perfect example of a sports icon. Why she not choose to play tennis masa budak dulu? If not, I pretty sure she will kick Sharapova's beautiful ass by now. Since person like her I bet she will excel in what ever field she chooses to involve with. She just knows the recipe for success.
Jimmy Choo (dia still ada share dalam company kasut tu ke?), Datuk Azhar, Leftenan Adnan dan ramai lagi contoh individu yang berjaya mencapai tahap tertinggi dalam bidang yang mereka ceburi. Everybody knows how important these icon to any society, right? They inspired us, they represent our country, they set a very high standard for us to follows and a lot more. Currently, I believe
We need:
1. World Class Performance Artist
2. Popstar Footballers
FAM conducts a sms contest last year to choose the most popular footballer for 2006. Tahu siapa menang? Syamsuri Mustafa, Terengganu's goalkeeper. Last year Terengganu not even play in MLeague. No offence to him, I believe cuma saudara mara and kenalan dia sahaja yang send those sms. Tidak pernah dalam sejarah bola antarabangsa pemain paling popular plays between the poles. But we just have no striker or midfielder yang cukup bagus to be promoted by local media. Akmal Rizal pernah memberi sedikit harapan, but anak emak tu rindukan sawah bendang masa main dengan club di Perancis. Khairy and Jason Lo pun masa create MyTeam, their only hope are to find 'Mawi' dalam dunia bola. So, I totally agree with them.
3. Gorgeous Female Celebrity Chef
Apart from Chef Wan yang hanya diminati oleh makcik-makcik, aku tidak kenal mana-mana chef yang glamer di Malaysia. Florence Tan did appear dalam Travel & Living beberapa kali, but auntie itu mungkin used to be gorgeous la kot. I hope to see female, Malaysian version of Oliver Twist (Naked Chef). Imagine
4. Flamboyant Billionaire
So, that's it. The top four icons that I hope to emerge from somewhere to inspire us.


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- #83 Betances, Canóvanas, Puerto Rico 00729
Business hours
- • Most insurance plans are accepted.
Products or Services
• It offers: physical therapy for children and adults
• Sports medicine injuries in athletes and visits
Payment methods
- Cash
R. S. Group Therapy provides physical therapy services for children and adults with different disabilities and health conditions. Home visits are made. Centre Multi-disciplinary therapy, attend adult and pediatric patients in occupational therapy, psychological therapy. speech pathology. 37 years of experience. Motor development, sports medicine, psychological assessment and treatment for children and adults.
The aims of physiotherapy depend on the condition being treated, while many factors influence the rate decision physiotherapy to use. In general all objectives are established according to increase or restore the ability of the patient's body or any part thereof for normal functional activities, and eliminate pain or discomfort that causes the injury.


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Some Freaks
In his debut film Some Freaks, writer/director Ian MacAllister-McDonald focuses on teenage misfits. Tagged as irredeemable outsiders in their Rhode Island high school, three seniors cope with indignation and hopelessness. Graduation brings the opportunity for re-invention, but playwright MacAllister-McDonald doesn't provide his characters with the self-awareness to discuss their futures: his pared-down dialogue is strictly present-tense.
He prefers to gaze at the shambling romance of introverted Matt Ledbetter (Thomas Mann), taunted for the patch covering a missing eye, and the brassy, overweight Jill Delise (Lily Mae Harrington). As they grow closer, Matt's twitchy, gay best friend, Elmo Moss (Ely Henry), who's related to Jill, feels increasingly left out. None of them can articulate their simmering emotions, and the charm of MacAllister-McDonald's casual observational approach wears thin.
Unspoken expectations and resentments boil over in predictable and dispiriting ways. Male characters are entrenched in cycles of self-pity: Matt defines himself through loss; Elmo continues to lust after a homophobic jock; and their golden boy classmate Patrick Keever (Lachlan Buchanan) recoils at the casual cruelty of his peers but does nothing about it.
The central story line involves Jill's drastic weight loss, and while Harrington's performance is a marvel – she conveys astuteness, resentment, triumph, and fear in a single expression – MacAllister-McDonald's exploration of her complex body issues is reductive and belittling. Jill's boisterous rebellion and sharp wit are presented as envy, and swapping her punk attire for blonde femininity is the grateful absorption of an outcast into the mainstream. It's her body, not herself.
Review by Serena Donadoni
Released on August 4, 2017 by Good Deed Entertainment
First published in LA Weekly, 2017.


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If you haven't heard by now Mr. Robert Williams B.K.A. Meek Mill has to appear in court over a probation violation, something the young men in our community know all too well. Apparently Meek failed to contact his probation officer to inform her that he was going out of town for work. Now before everyone goes into a tizzy yes Meek's P.O. knows he's an entertainer but she cannot just allow him to come and go as he pleases. It's the same procedure for everyone in the commonwealth of Pennsylvania. For all we know Meek just forgot or assumed that someone on his management team had taken care of it. It could be a simple oversight or it could be that Meek was feeling himself and figured he'd be alright even if he didn't call his probation officer. It's funny because some kids out here really believe that being famous can get you out of a bad situation.
If that were true none of our favorite emcees would have ever done any time! Tupac, Capone, Lil Wayne, Snoop Dogg, Shyne, Cassidy, Remy Ma, and everybody else I didn't mention. These emcees got knocked at the height of their career or right before their careers were to sky rocket. It's almost like when a close friend or family member was about to turn their life around but end of getting killed. When a celebrity goes to jail or prison they are dead to the industry in a sense. Unless you are a Tupac or a Snoop Dogg nobody is going to be checking for you when you're released. Remy survived off her husband and bars alone! Celebrity saves no one and Meek Mill may soon remember what that feels like since he was just released. Word is that Nicki Minaj has or at least plans to make a plea to the judge for her beau. Now tell me this, if they were "the average" couple would she really go out of her way to do so? Half the girls out here are sending their boo to jail over some BS. I'm a woman so don't misconstrue the point I'm trying to make. She didn't do it the last time but I guess that's because she was in the middle of breaking up with Safaree. (Insert any shade emoji you'd like) The question is why is Nicki using her celebrity to appeal to the judge for Meek Mill? Why not? If you could you would. That doesn't mean it's going to work.
So, if Meek goes back to jail then what? WWND? What will happen to the remainder of his fan base that didn't side with Drake? You will see memes of Meek in jail while Drake makes his move for Nicki. That was just a joke but that's the day and age we live in if it's a meme than its major. This is just a word to the wise that being a celebrity or having money doesn't work for melanin people and barely works for someone the system sees as poor which is me and all of you. You are not immune to the woes of the world just because a few million people know your name. For people in our community there's no such thing as n***a we made it.


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The world is getting attracted to wireless technology. Wireless Technology helps you to get rid of the mess that is created with the cables and makes your computing at your own comfort. It is widely becoming a household norm and with advanced technology, you can even improve the connection of wireless connections and make your work more comfortable.
Here Are Ways to Improve Wireless Connectivity
1. Check The Router Position
You need to check the position of your router. The wireless signal cannot reach if your router is not positioned well. For this, you need to ensure to centrally locate the router in the home. So that the best range of signals is possible. Ensure this by placing the router away from the obstructions on a flat level surface.
Also, to improve the connectivity of your wireless connection, you must also ensure that you are using a wireless channel that is unique as this helps in limiting the interference that may be caused by neighboring wireless signals.
2. Try to Replace Antenna
Usually, routers are shipped with smaller antennas that are of Omni-directional potential. These antennas transmit the signals in all the directions which are quite useful if you prefer to use wireless throughout the house. But the disadvantage that is associated with this kind of antenna is the range of signals it transmits that are quite short.
So to improve the performance of your wireless connectivity, make sure to replace the antennas with a directional antenna. This kind of antenna is capable of improving the range of signals by focusing on a specific way and thereby allows you in aiming where it is actually needed. These are famous as "high-gain" antennas and signals increase can be calculated in decibels (db).
3. Invest In A Repeater
One of the best and safest ways to boost the signals of your wireless connectivity is by using a wireless repeater. The repeater works similar to a router. But it does not create any signal.
It focuses on existing signals. Installing a repeater is quite easy as it does not involve any mess of any additional connections or wires. You can use multiple repeaters for creating a home or network of business with comprehensive connectivity.
4. Firmware Update Of Router
Home users often overlook the importance of firmware updating of the router. Business networks usually get some notification regarding these updates. In order to improve the connectivity of your wireless network, check for the firmware updates of your router.
You can find a section in the interface of your router for firmware updates. Frequently check the website for the updates offered by the manufacturer of your router. You can upload these updates with the help of the router's interface.
5. Prefer Power Line Network
We recommend you to consider powerline adapters as they help in creating a home network that is very fast by using electrical cabling in the house. By doing this, you can work from any part of your house without compromising the connectivity performance. It is quite easy to create a powerline network. For this, you need to plug in a power socket close to your router. Now, connect the router by using Ethernet cable.
Ethernet cable is usually supplied along with the adapter. Now, you can connect the second adapter into a power socket and you can connect this to your laptop, smart TV using another Ethernet cable. Powerline adapters perform well as if they are directly connected to the router. In fact, they are actually plugged in other parts of your house.


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24 Jan 5 Marketing Campaigns we Loved in 2018
Let's admit it, 2018 was a whirlwind. From Prince Harry getting married to the Laurel and Yanny debate (we're at three Yannys and one who plugged her ears and refused to listen) the Winter Olympics, and the false missile alarm in Hawaii, it was hard to keep up!
There were so many new and exciting marketing campaigns in 2018 that we just had to take one more look. From inspiring to attention-grabbing to funny, check out the 2018 marketing campaigns that made our top five list.
Five marketing campaigns that made us laugh, cry, and most importantly, TALK.
- Grabbing a controversial subject by the horns
The Nike® – 'Colin Kaepernick – Just Do It' ad may have easily been the most controversial ad of the year. When I first saw the ad on social media, I was surprised. It featured a photo of Colin Kaepernick's face with the statement, "Believe in something. Even if it means sacrificing everything." Nike made a huge decision to have Kaepernick, the first football player to kneel during the national anthem, star in one of its ads. After all, taking a knee during the Star-Spangled Banner—and the reason behind it: supporting the #BlackLivesMatter movement protesting police brutality against the black community—has been a heated debate that's divided the country since 2013. Didn't Nike fear losing a huge segment of its consumers?
Turns out, Nike conducted research and found that its target audience, 18 to 34-year-olds, believed that companies should take a political stance and they supported players who kneeled during the national anthem.
The results of the ad? A LOT of social chatter, chaos, media attention, oh, and a healthy boost in sales.
The ad resulted in a surge of Facebook and Twitter shares, and a whopping 170,000 new Instagram followers around the debut. The ad also led to a host of videos featuring people burning their Nike apparel, or pledging to never buy another Nike product. Amidst all the chaos, Nike emerged a winner because the people who were upset weren't the majority of their target audience. Rather, the marketing campaign resulted in a 31% increase in online sales.
This Nike ad is a great example of how conducting research and narrowing in on your target audience can land you one of the best marketing campaigns of the year—and get people talking for months.
- The perfect response to a chicken shortage crisis
Did you say a chicken shortage? Last February, KFC® had to shut down many of their restaurants in the UK because they ran out of their key product: chicken. How that happened is a different story, but the chicken shortage frustrated many of their consumers (I'd be upset if I couldn't get my chicken fix, too).
While a chicken shortage is no laughing matter, it's also not a world crisis. KFC had to quickly find the appropriate response to the situation. In lieu of a serious apology for running out of chicken, KFC opted for a wittier response. KFC rearranged the letters in their logo to say "FCK" in a print advertisement as a way to say sorry for closing many of its restaurants' doors. Since the f-word is not considered as offensive in the UK as it is in America, it was just the right response to the chicken shortage. The witty advertisement not only helped KFC, but its index score jumped from -10.6 to -0.7 (higher than what it was before the chicken crisis). We can all learn a lesson from KFC: know your audience to appropriately respond to a crisis.
- Wait, you changed your name for burgers?
Dear Internet, we abbreciate your batience. Now let's see who guessed right. B-hold!!!!! #IHOb pic.twitter.com/Fh3SkZ7s3Y
— IHOP (@IHOP) June 11, 2018
One day I was scrolling through my Twitter newsfeed when I came across an article: IHOP® was changing its name to IHOb. Intrigued, I clicked on the article to learn what the "B" in IHOb meant. After all, isn't IHOP known for pancakes? Why would they want to change it? I learned that IHOP was changing its name to "International House of Burgers" to promote itself as more than just a breakfast restaurant (a name change ruse they kept up for a few days).
Want an effective advertising campaign? Get creative and take risks, just like IHOP. The company used social media marketing to announce its name change to IHOb, a small notion for a company seemingly changing everything it stood for by denouncing pancakes and jumping on the burger bandwagon. IHOP got EVERYONE on social media talking about them after they saw the temporary name change and watched IHOP's burger promo video. I'd say they achieved their goal of increasing awareness that they sell more than just delicious pancakes. Great job on the publicity stunt IHOP (or should we say IHOb?)!
- No, really, "It's a Tide Ad"
If you didn't tune in to last year's Big Game, you missed out on one of the most unique and entertaining Tide® commercials. You start out thinking it's just another Old Spice commercial, but then near the end they announce, "It's a Tide Ad." From one funny scene to the next of popular commercial reenactments, it was hard to stop watching! It also featured David Harbour, the famous Jim Hopper on the Netflix Original series, Stranger Things. Now who could resist that?
Almost everyone knows Tide, the famous laundry detergent, so why spend the big bucks to play an advertisement during the Big Game? That's easy. The main goal of Tide's "It's a Tide Ad" marketing campaign was to increase brand likability through familiarity (this is why they used David Harbour) and humor. Tide was in a negative spotlight for a while after kids got sick from eating Tide pods (not something they would ever promote, BTW), so this was a smart marketing move on its part. The company's creative video marketing had people laughing and talking positively about the brand, making it worth the expense of that prime-time slot.
- "Dilly Dilly"
"Dilly dilly" could have easily been the catchphrase of the year, which is why we've also chosen Bud Light®'s "Dilly Dilly" advertising campaign as one of our favorite marketing campaigns of 2018. While the true meaning of the phrase is unknown (some speculate it means "Cheers"), not laughing at Bud Light's "Dilly Dilly" advertisements during the Big Game (or any of its previous Dilly Dilly ads) was a nearly impossible task.
If you ever need help brainstorming a rallying cry that will increase consumer engagement and sales for your business, consider asking Bud Light! Just like the Budweiser® Frogs or the Budweiser "Wassup" commercials, Bud Light's Dilly Dilly ads do an excellent job of getting people to repeat their favorite commercials willingly! Now when consumers take a trip to the grocery store, they can't help but whisper "dilly dilly" as they walk past the Bud Light cases. If you want to test out your rallying cry to see if it's as successful as Bud Light's Dilly Dilly, see if it makes the list of banned exclamations during the next golf tournament at the Augusta National Golf Club.
Good marketing campaigns have these things in common: they tell a story (even if it is just a spoof, like IHOb), connect with the audience on an emotional level (like Nike's controversial ad featuring Colin Kaepernick) and deliver on a key insight (like IHOP's ad that addressed what we can only guess was a pain point with consumers: I want more options than just pancakes when dining out). When brands can achieve all three, they might just be lucky enough to make 'best-of' lists like this one for years to come.
Ciara is a senior at the University of Wisconsin Oshkosh majoring in Interactive Web Management. Her major covers a wide array of subjects, including journalism, marketing, information systems and computer science. Through her class and internship experiences, she has developed her graphic design skills by creating content in Adobe Photoshop for online and print use. She is also familiar with researching and implementing social media best practices, has an understanding of HTML and CSS, and enjoys writing blog posts on various topics. As an animal lover, Ciara loves volunteering as the marketing coordinator for the Winnebago Pet Expo, a nonprofit dedicated to building dog parks in Winnebago County.