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Sensors 2012, 12(9), 12710-12728; doi:10.3390/s120912710
Abstract: Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR) chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear). Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.
1. Why Is Candidate Biomarker Validation Important?
Validation of candidate biomarkers has a single goal: to determine, if there is sufficient evidence for a potential clinical utility of a given biomarker candidate to ensure further investment in that candidate for clinical trials. A validation is therefore essential before moving forward with this potential biomarker candidate. Thus, a clinical study is expensive, time-consuming, and has a high demand for clinical samples. Recently, a profound biomarker evaluation ensured the quality and clinical validity of a diagnostic assay . Paulovich et al. dubbed biomarker validation as the tar pit, since the majority of biomarker candidates failed the clinical phase. Hence, the success rate of implementing a new biomarker candidate into clinical use is extremely low. However, plenty of publications about new biomarkers and diagnostic test platforms have been released within recent years. But, as the study of Fontela et al. revealed, published diagnostic tests for infection diseases often miss methodological quality and accurate reporting . They used Quality Assessment of Diagnostic Accuracy Studies (QUADAS) and Standards for the Reporting of Diagnostic accuracy studies (STARD) tools for improving the reporting of diagnostic accuracy assays, regarding the quality of diagnostic studies in tuberculosis, malaria and HIV. Through systematic search of literature using PubMed and EMBASE (2004–2006), the sensitivity and specificity of various commercially available tests was compiled. Based on different quality items, they concluded that all studies had design deficiencies. For instance, merely 10% of the studies had an adequate description of the reference standard, less than 25% of the studies included a description of withdrawal, and none of these studies reported methods for the calculation and estimation of reproducibility.
Insufficient diagnostic test accuracy is just the tip of the iceberg. It is the consequence of poorly designed biomarker candidate discovery and validation phases without clear understanding of the nuances of interpreting high dimensional data sets which often leads to biases and high false discovery rates . Many of these large list candidate biomarkers may discriminate between two classes of interest, but do not conform to the high standards of clinical trials. Moreover, candidate biomarkers that display the most significant differences between the cases and control group in the discovery dataset are often preferred without being tested, whether or not these are the most beneficial analytes for clinical decision making. Strategies which allow a high number of candidate biomarkers to be analyzed with the highest throughput and the lowest possible costs are required for an impartial validation. Based on the circulatory nature of blood through almost every parts of the human body, the measurements of blood components are particularly valuable for monitoring the health state of a person . Certainly, proteomic-based biomarker discovery and validation directly in serum is challenging due to the complexity and the dynamic range of the analytes in plasma. The concentration range of serum proteins extends through eleven orders of magnitude, from albumin through cytokines . Matters are complicated by the fact that potential candidate biomarkers are often present in low concentrations and are often bound to carrier molecules. Consequently, predominant high-abundance serum proteins may interfere and considerably influence the assay performance, as well as the quality of the result. This so called serum matrix effect, broadly defined as interference with the analytical technique by one or more components of the sample, can lead to loss of assay robustness, sensitivity and high levels of false positive and negative results .
The matrix effect is exceedingly relevant for mass spectrometry (MS) analysis, one of the principal enabling technologies for unbiased identification of novel target antigens. The milestone paper, that cemented the role of MS in clinical proteomics, was published by Petricoin et al. They identified components of the serum proteome by MS that allow a differentiation between patients with ovarian cancer from healthy individuals . Numerous papers have been published that aimed to completely discover novel target antigens for diagnostic application by the help of MS [8–10]. However, insufficient analytical sensitivity of MS complicates the detection of low-concentration biomarkers within a complex mixture of high-abundance proteins . Overcoming these detection limits is the biggest hurdle in MS analysis. Hence, the depletion of abundant protein fraction and the enrichment of biomarker are imperative to increase the sensitivity of the subsequent MS analysis . Apart from the highly-abundant serum proteome, the discovery efforts alternatively focus on tissue or body fluids and moving to plasma once candidate biomarkers have been identified. Indeed, the protein composition of serum differs from that of tissue or body fluids. Serum protein depletion is a very time-consuming process, but the most critical issue is the accuracy and reproducibility of the analytical process caused by software problems. To increase the analytical reproducibility, MS labs typically run their samples 10 or more times, which implies a consumption of precious patient sample . MS data helps to identify novel target antigens, but neither contains information about the applicability of the candidate in an immunoassay, nor the clinical validity.
A further strategy to identify novel antigens is via phage, bacterial, or mammalian cell-based cDNA libraries . An important limitation of this approach is the lack of post-translational modifications, including glycosylation and phosphorylation. However, post-translational modified target antigens often contribute to diagnosis of autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus [15,16]. Post-translational glycosylation of antigens also represents an important target of anti-viral responses against HIV and influenza .
The current gold standard for biomarker validation and clinical diagnostics is the classical ELISA. Since this technique allows a relatively high-throughput and is a versatile and robust tool, ELISA is constantly employed for confirmatory studies. Thus, microarray analysis is often matched against the quantitative data of ELISA assays . However, the ELISA is limited by the fact that the classic approach allows only a single antigen detection and often requires relatively large volumes of sample material compared with state-of-the-art methods. This concern becomes especially acute when clinical studies require access to limited amounts of biological material. Unfortunately, the ELISA performance strongly depends on the antibody quality as well as on the operator's skills and experiences. The solid nature of ELISA results in altering the immobilization of antigen to the solid matrix, which causes problems associated with accuracy and reproducibility. In comparison to other multiplexed immunoassays, ELISA has a relatively narrow dynamic range. This becomes particularly relevant when the biomarker of interest is detected in serum. Differences between samples are biased by comparison of serum that has biomarker levels within the dynamic range (does not require dilution in the assay) to serum above the dynamic range (does require dilution). The development of an ELISA assay is costly (typically, US$100,000–US$2 million per biomarker candidate) and is associated with a long development lead time (>1 year) and a high failure rate . Compared with MS and traditional ELISA, multiplex arrays have several advantages, including their high-throughput nature, requirement for smaller sample volume, efficiency in terms of time and cost, the ability to evaluate one antigen in the context of multiple others, and the ability to reliably detect different proteins across a broad dynamic range . Multi-analyte immunoassays might be the tool of choice for the identification and detailed validation of protein biomarkers in serum.
2. Multi-Analyte Immunoassays
Today, immunoassays are the fundamental method of protein analysis and have gained importance as powerful tools in basic and applied proteomic research. Immunoassays have their origin in the 1950s, when Berson and Yalow developed the first immunoassay for insulin, using radioactive labeled antibodies for detection . To avoid radioactivity, in 1971 Engvall and Perlmann introduced an alkaline phosphatase conjugated antibody for detection that displays the classical detection principle of an ELISA (Enzyme-linked immunosorbent assay) . In the late 1980s, Ekins, the founder of multi-analyte immunoassays, conceived the “Ambient-Analyte-Theory” and discussed the application of a parallelized microspot immunoassay in immune diagnostics [23,24]. His theoretical examinations and his experimental work demonstrated that simultaneous microspot immunoassays can be carried out with classical ELISA. Through miniaturization, a greater sensitivity and selectivity, as well as a higher throughput can be achieved. However, Ekins' theory gained even more importance after the development of DNA chip technology in the 90's, which coincided with the worldwide breakthrough of biochip technology . The innovation of advanced equipment such as pipette robots, spotters and fluorescence readers allowed the production of microarrays with thousands of microspots, as well as the sensitive and high-resolution detection of bound target molecules. Since DNA microarrays are a well established tool in molecular biology, several groups have used this technology in order to immobilize proteins instead of DNA on the arrays [26,27]. Those arrays are miniaturized immunoassays used for protein or antibody detection from complex media. The rapid rise of immunoassays in protein analytics, combined with the development of protein arrays, and later peptide arrays as multiparametric immunoassay, illustrates the enormous potential of this technique in diagnostics. Within the last decade, multi-analyte immunoassays have led to the identification of a huge number of biomarkers from serum, particularly new antigens, and to the elucidation of disease progress. However, the current limitation is the validation of biomarker candidates. One of the major challenges in biomarker validation and diagnostic assay development is the availability of high-quality capture molecules, e.g., antibodies. Several efforts have been seek for the assortment of well-characterized affinity-based molecules, not limited to antibodies, in one database [28,29]. But also different other aspects of the entire validation process need to be improved. Technologies like MS and ELISA currently used for biomarker discovery and validation are not able to achieve the combination of high throughput with high measurement accuracy and precision. Compared with traditional ELISA, multiplex arrays have several advantages, including their high-throughput nature, requirement for smaller sample volume, efficiency in terms of time and cost, the ability to evaluate one antigen in the context of multiple others and the ability to reliably detect different proteins across a broad dynamic range. Multi-analyte immunoassays are excellently suited for the entire biomarker discovery process. During the validation procedure biomarkers must be identified and the disease association must be explained as well as the assay conditions must be defined before moving with that candidate in clinical trial (Figure 1). Hence, a clinical study is expensive, time-consuming, and a consumption of rare patient samples should be prevented. The group of Peter Nilsson pointed out the potential of staged proteomic profiling of serum by using a combination of different affinity-based methods. They had also considered the primary problem of biomarker analysis: the establishment of efficient throughput methods for validation of large generated datasets. Larger studies are needed to investigate the protein expression with the help of independent microarray platforms, and to finally overcome the inherent throughput limitations of Western Blot and ELISA. This dual confirmatory approach allows large-scale cross platform analysis and validation of antibody performance under assay conditions .
The evaluation of clinical validity and utility is imperatively needed. The process of identification and validation of serological markers as well as their application in diagnostic routine requires a combination of independent immunoassay platforms with the possibility of high-throughput measurements. This paper focuses on microarrays, bead assays and array-based surface plasmon resonance (SPR) assays and discusses their application in serum biomarker discovery.
The process of candidate biomarker validation has to be improved by using an approach comprising different affinity-based methods with the possibility for high-throughput. Rapid screening of interactions and biomarker disease association is carried out with microarrays. Label-free methods like surface plasmon resonance (SPR) are strongly recommended for the in-depth validation of these interactions. This analysis provides data about association and dissociation phases, as well as affinity constants. With the help of an affinity ranking, the interactions are classified according to their properties. Kinetic parameters and the kinetic classification allow the development of fine-tuned diagnostic assays concerning incubation time and washing stringency. Both strongly contribute to the implementation of candidate biomarkers into a new diagnostic assay. The defined assay conditions and performance of the candidate biomarkers can be tested in a bead assay.
3. A Cross Platform Comparison
Microarrays are miniaturized biological devices within which capture probes are fixed in defined positions on a supporting material, normally a standard sized glass slide. The analyte bound to the capture probe is detected by fluorescence. Microarrays made Ekin's concept of a miniaturized bioanalytical assays a reality. They allow many parameters to be tested at the same time under different condition. With parallelization in mind, the microarray can be divided into several subarrays. This makes the parallel screening of thousands of capture probes in one experiment possible. Such microarrays are ideally suited for an unbiased and rapid identification of a host of antibody-antigen interactions. However, problems including immobilization efficiency, fluorescence detection, data analysis, and signal-to-noise ratio (S/N ratio) must be critically addressed to improve the assay performance .
Glass slides often function as solid supports on which thousands of different capture molecules can be immobilized. Since tiny amounts of sample volume (reaching up to several nanoliters) per microspot are deposited, special liquid handling robots are required. Those are often constructed for the purpose of the DNA microarray development. Nevertheless, the spotting of purified proteins or peptides is much more challenging. They are more chemically diverse than DNA and their stability and functional nature on the array have to be ensured. An alternate approach to spotted proteins is the Nucleic Acid Programmable Protein Array (NAPPA) that circumvents the technical limitations of protein spotting. It is constructed by spotting protein-encoding plasmid DNA on an array surface and the proteins are subsequently generated using cell-free expression systems. Thus, the NAPPA platform allows functional protein studies including protein-small molecule, protein-protein, protein-nucleic acid, and also antigen-antibody interactions . NAPPA microarrays were used to detect autoantibodies in human sera of breast cancer patients to p53 antigen and to GAD65 antigen in type 1 diabetes patients . Dependent on the microarray application, several covalent or non-covalent immobilization strategies for capture probe binding are established (Table 1). These microarray substrates have to fulfill two major requirements to achieve maximal immobilization efficiency: It is important that the substrates provide high binding capacity and preserve the capture probe in an active state .
On aldehyde, epoxy or amine coated substrates every functional group of the protein can react with the reactive group on the surface, therefore proteins and peptides are immobilized randomly. Thus, chemically or biologically active domains are sometimes not accessible for the analyte. For directional immobilization, the capture probe has to be chemically functionalized to differentiate the domain available for immobilization from those of chemical/biological activity. This leads to higher signal intensities and improved S/N ratios . Schulze et al. demonstrated that special amino acids such as histidine and tyrosine at the N-terminus of the capture probes resulted in an improved immobilization efficiency on epoxysilane surfaces and higher signal intensities . Ethanolamine that reacts with the epoxy group and does not preferably cover the capture probe has been used as blocking solution for epoxy surfaces. Thus, the S/N ration is additionally increased. Epoxy and other functional group substrates constitute two-dimensional coatings since the capture probe is in direct contact with the activated glass surface. Alternatively to epoxy, the capture probe can also be a part of simple monolayer architecture. Andresen et al. prepared a monolayer mixture of streptavidin and biotinylated peptides for site-specific immobilization on aldehyde coated glass slides in a two-step procedure (Figure 2B) . This robust peptide microarray platform allows serological diagnosis of infection diseases. Beside these two-dimensional surfaces, three-dimensional coatings were developed as well (Figure 2D). The three-dimensional space of these polymer-based hydrogels leads to a higher capacity for capture probes. More molecules can be immobilized onto a limited space . Higher amounts of immobilized capture probes results in increased total signal intensities and assay sensitivity. Since polymers self-adsorb onto the glass surface when simply immersed in aqueous solution, the coating procedure is fast and inexpensive. To improve the microarray robustness and reproducibility, Schröder et al. focused on the implementation of dual-color read-out for antibody microarrays . Protein samples such as plasma, serum, urine, tissue, or cell culture can be analyzed without prior expensive and elaborate protein depletion steps. Depending on the study, there are two different experimental design options. For a direct comparison, two different sample types are labeled with different fluorescent dyes and competitively incubated on the same array. As an alternative a reference-based design is possible. Thereby, all protein samples are labeled with the same fluorescent dye and competitively incubated with a common reference, which is labeled with the second fluorescent dye. The evaluation of signal intensities is carried out by using the ratio of the two color channels for identification of differences between the samples. Technical variation effects are abolished by considering the ratio of the signal intensities. This leads to higher reproducible data, sensitivity, and consequently improved assay robustness. Overcoming the practical hurdles, microarrays represent a sensitive screening tool for serum biomarker discovery and have the capacity for an unbiased approach. Analyzing the IgE reactivity in patient sera, Hiller et al. used a microarray composed of 94 immobilized purified allergen molecules that represent the most common allergen sources . This allergen microarray allowed the determination and monitoring of patients' IgE reactivity profiles and the identification of allergy eliciting molecules. In order to characterize the antibody response of distinct human autoimmune disorders including systemic lupus erythematosus and rheumatoid arthritis, Robinson et al. screened different patient sera by the help of peptide microarrays . Thereby, the specificity and pathogeneses of autoantibodies was elucidated and new autoantigens were identified. In serum diagnostics, peptide arrays are well-established tools for antibody response screening. Peptides are only present as continuous epitopes. Nevertheless, peptides have many advantages compared to native protein antigens . The synthesis of peptides is chemically established and completely automated. Based on their short length (10–15 amino acids), peptides hardly build up a secondary or tertiary structure which makes them chemically and physically more robust than proteins. Furthermore, diverse covalent and non-covalent immobilization strategies of peptides, up to the direct synthesis on solid support, are known and established . In addition to their application as screening tools of autoimmune disorders, peptide microarrays have been successfully used for the detection of microbial infection diseases. Andresen et al. developed a peptide microarray consisting of viral peptides to demonstrate the specific binding of monoclonal antibodies . In this regard, they were able to detect the binding of commercial available antibodies up to picomolar concentrations. That demonstrates the potential of microarrays, screening for antibody subtypes which are usually present in low concentrations like IgE. Similarly, bacterial infections like tuberculosis caused by Mycobacterium tuberculosis can be diagnosed. Using peptide microarrays, Nahtman et al. identified novel antibody epitopes and characterized the antibody reactivity profile on bacterial antigens . However, if there is a lack of known antigens, random sequence peptide libraries can be used for serum screening . In this manner, the antibody repertoire binding pattern of two different mice strain sera—healthy and infected with Heligmosomoides polygyrus, an intestinal helminthic parasite—were analyzed. Due to the library design and sophisticated microarray data analysis, a classification between healthy and infected mice could be predicted with a small number of peptides. Besides planar microarrays, robust and flexible bead assay systems have been developed over the last decade. The design of a bead-based immunoassay is comparable to that of a microarray: the capture probes are likewise immobilized on a solid support—beads. In this case, the capture probe is bound to microspheres and incubated with the analyte in suspension, which allows an improved mixing procedure and automation (Figure 2C).
These microspheres can consist of a variety of different materials, like color-coded beads or magnetic particles with different diameters. In comparison to location coded microarrays, whose capture probes are printed in picoliter volume pointwise onto the supporting material, a liquid handling robot is not imperatively needed for bead assays. Since bead assays are usually performed by using standard laboratory equipment, i.e., using microtiter plates and a flow cytometer. The individual capture probes on the microarrays are detected as illuminated spots, depending on their location on the array surface. In the case of bead-based systems, the differentiation of capture molecules occurs with a color-coding of the beads. The color coding of the beads offers the potential of multiplexing. Depending on the number of different colors, each bead population can be coated with another capture probe that allows the binding and detection of specific analytes from a serum sample. A mixture of color-coded beads that have bound the antigen are incubated with a serum sample in a cavity of a microtiter plate (Figure 3). Serum antibodies with the corresponding paratope recognize and bind to the antigen. The higher the concentration of corresponding antibodies in the sample the more antibodies can bind to the beads. The detection of the bead-bound antibody-antigen-complex occurs with help of secondary fluorescent-conjugated antibodies. The spectral range of the antibody fluorescent dye differs from this of the internal microsphere dye. Thus, the classification of the beads (red, green) and the quantification of the signal intensities caused by the detection of antibody-antigen complex (blue) can be simultaneously read out by a flow cytometer. The outcome of the multiplex antibody-antigen reactions are displayed fully automated in the analysis software. The Luminex xMAP technology has become the most popular bead assay platform and belongs to one of five assay platforms available to serologically diagnose the West Nile Virus infection . This bead assay consists of 100 different bead populations, each of them with two fluorescence dyes in 100 different coding ratios which are used for multiplexing. Via the fluorescence coding of the bead populations, a specific labeling is assigned for every immobilized molecule species.
Another platform, the BD FACSArray Bioanalyzer, is based on flow cytometry measurements. The differentiation between different bead populations occurs over a range of bead sizes and two fluorophores, which increases the degree of assay complexity. Bead assays enable the analysis of different capture probes within one reaction tube such as the cavity of a microtiter plate. In clinical diagnostics, these plates have become a usual lab standard and allow an automated high-throughput treatment of patient samples. Due to the easy handling and flexibility of the assay conditions, many commercially available immunoassays for the detection of allergies, autoimmune diseases, and infections are bead based. Earle et al. developed a fluorescent multiplex array for detecting six common indoor allergens in house dust samples . This assay consisted of monoclonal antibodies that were covalently coupled to fluorescent microspheres of the Luminex xMAP platform. These antibodies have been well-defined and have a high specificity for their epitopes, a fundamental requirement for developing a reliable assay. The same group also studied intensely the crystal structures of allergens, as well as the interaction between these allergens with their antibodies [50–53]. The assay performance, including sensitivity, detection limit, and reproducibility was compared with an ELISA method, the gold standard for environmental exposure assessment. The determined intra-assay reproducibility resulted in a CV of less than 10% and in most cases the inter-assay CVs of the multiplex bead assay (>15%) were found to be slightly better than that of a normal ELISA. By doing a multiplex bead assay the dynamic range of the measurement was increased and allowed each sample to be tested at only two dilutions (1:100 and 1:10,000) to cover the full range of allergens. Compared to normal ELISA the detection limit was an order of magnitude lower for the bead assay and decreased to less than 1 ng/mL for each of the allergens. In this regard, a greater assay efficiency and reproducibility was achieved by measuring six or more allergens simultaneously in a single microtiter well. This has led to several technical improvements compared to normal ELISA. Currently, the presented multiplex array is commercially produced and sold by the company Indoor Biotechnology Inc. The number of measured antigens has been increased allowing the simultaneous detection and quantification of eight common indoor allergens in house dust samples. This technology represents the state-of-the-art technology in allergen detection.
Abreu et al. used the FIDIS™ technology by Biomedical Diagnostics for simultaneously detection of autoantibodies to diagnose the chronic autoimmune disease rheumatoid arthritis . The FIDIS™ technology combines the Luminex xMAP® platform with a flow cytometer, and includes a software for data analysis. This offers technological opportunity in laboratory bench top integration and automation. The antigens, human and animal IgG Fc fragments, were covalently bound to different sets of microspheres and incubated with patient serum samples. The autoantibody levels, measured with FIDIS™, were compared with routine laboratory tests regarding the clinical sensitivity and specificity. The results demonstrated the efficiency of FIDIS™ with an analytical performance equivalent to conventional methods such as ELISA and latex agglutination test. Nevertheless, the important benefits of FIDIS™ are the time and cost-saving measurements and the use of minimal sample volumes.
Bead assays are also used for the detection of arising infections. Binnicker et al. applied the BioPlex 2200 system that was developed by Bio-Rad Laboratories in cooperation with Luminex for the serological diagnosis of primary acute Epstein-Barr virus (EBV) disease . The EBV diagnosis requires a combination of several antibodies for a reliable prediction. Thus, multiplex immunoassays represent an appropriate technology for identification of multiple antibody populations in a single reaction and thereby expediting detection of EBV. The serological assay was based on the simultaneous detection of five different IgG and IgM class antigens. The EBV viral capsid antigen (VCA), nuclear antigen-1 (NA), and early antigen-diffuse (EA-D) represent IgG epitopes, whereas heterophile antibodies and IgM-VCA were IgM derived. The serological response to EBV, obtained with the BioPlex 2200 system, was comparable with the results of conventional methods used to detect EBV specific antibodies. Thus, the assay yielded the same sensitivity and specificity as routine tests such as indirect immunofluorescence assay and enzyme immunoassay. However, the interpretation of data, and a confidential diagnosis appears to be crucial, and several groups have dealt with this issue. In conclusion, BioPlex 2200 EBV assay represents a valid evidence-based tool but, should be used in association with other laboratory and clinical parameters for classification of EBV disease state [56,57].
Similar to microarrays, the surface coating of the beads and appropriate blocking is important for assay performance. A major drawback of bead assays is that human serum components can directly bind to the beads and cause unspecific background. Serum pre-incubation with polyvinylalcohol, polyvinylpyrrolidone, and other proprietary reagents like Super ChemiBlock and Chemicon could reduce unspecific binding and resulted in increased S/N ratios . Compared to planar microarrays, bead assays are not suited for the identification process of novel serum biomarkers due to the limited capture probe capacity. In a single microarray experiment up to 10,000 interactions can be simultaneously analyzed compared to bead assay with up to 500. However, bead assays are a versatile tool for routine application due to their easy automation and flexibility in experimental conditions. Novel serum biomarkers and predefined assay conditions can be easily implemented in a bead assay platform to determine the fine tuning of assay development.
The presented immunoassay platforms-microarrays and bead assays-enable a high throughput analysis with tiny amounts of patient sample. These endpoint measurements allow a yes/no answer in terms of analyte binding to the capture probe. However, the interplay between molecules in vivo is dynamic and does not follow a steady-state-equilibrium or adhere to a binding model. Therefore, a kinetic analysis that provides parameters like association and dissociation rates, as well as equilibrium constants, is sorely needed. These studies are of profound importance regarding the development of a diagnostic assay. Especially useful for optimizing the assay conditions, kinetic parameters are an indication helpful in defining the duration of incubation time and the washing stringency. Both parameters contribute to an improved assay performance while minimizing background noise, as well as non-specific binding to the target molecules. The kinetic measurement of antigen binding to antibody microspots on an array has been discussed by several reports. The conclusion of these reports is that mass transport dependency of the antibody-antigen microspot kinetic has been one of the main restrictions of array technology . Although, the effects of microarray design parameters on microspot kinetics were theoretically and experimentally analyzed, the mass transport limitation still remains a serious problem. The optimization of parameters such as incubation vessel geometry, incubation time, stirring, capture probe density, spotting pattern, analyte concentration, etc. led to an improved sensitivity of kinetic microarrays. Nevertheless, mass transport limitations could not be entirely prevented to generate confidential kinetic parameters of the interaction process .
SPR is a powerful technology to study label-free antibody-antigen interactions in real time. Particularly, high-content microarray-based biosensors are well suited for diagnostic screening of serum samples, epitope mapping and protein expression profiling . The SPR phenomenon occurs when polarized light passes the interface of a high-refractive-index material (prism or grating) and a low-refractive-index medium (analyte). The light beam is completely reflected by a thin gold film. Another component, called the evanescence wave, passes into the gold film, where plasmons are excited. Thus, a resonant wave is transmitted and the intensity of the reflected light is decreased. Mass absorption by analyte binding to the capture probes on the gold surface leads to local refractive index changes. This causes a change in the SPR angle which is visualized as Response Units (RU). The progress of interaction is displayed in a sensorgram, a plot of response units against time (Figure 4). Conventional SPR systems have a few flow chambers, a maximum of four to six. As a result, only a limited number of analytes can be simultaneously measured. Serum screening is only possible if a small set of antigens is used. Nagel et al. restricted their SPR studies for serological detection of Lyme borrelioses to two widely used antigens. The whole proteins as well as two peptides, representing immunodominant domains, were used as capture probes . But in general, different flow chambers have different antigens, and a direct comparison of kinetics and affinity ranking of more than four interactions is not practicable. De Boer et al. used a SPR platform that combines the microarray principle with SPR detection in one flow chamber. Thus, all SPR analyses were performed on a single array. The microarray contained 144 different glycans derived from the human parasite Shistosoma mansoni and was used for the simultaneous detection of glycan-specific serum antibodies . Differences in the anti-glycan antibody repertoire from sera of S. mansoni infected patients, as well as uninfected controls were monitored. Moreover, differences between the antibody classes that responded to the infection were also revealed. A SPR biosensor was used to detect antibodies directly from human blood serum against the immunoreactive peptide epitope of EBV nuclear antigen. The results, yielded with this biosensor, were characterized in terms of reproducibility, detection limit and regeneration and compared with a conventionally used ELISA . The reproducibility of the SPR sensor was obtained by using the standard deviation of the sensor response of different sensor chips and was determined in the range of 8 and 18% dependent on the serum antibody concentration. For the corresponding ELISA the inter-assay standard deviation was found to be between 5 and 20%. Thus, the reproducibility of the SPR sensor is comparable to that of an ELISA. The detection limit was estimated to be 0.1 ng/mL, which is lower by an order of magnitude than the detection limit of ELISA. The regeneration procedure was optimized and a minor loss of sensitivity was observed after 10 measurement cycles. This demonstrated the clinical relevance and the potential of the SPR sensor for usage in EBV diagnostics.
Non-specific adsorption of proteins from complex media such as serum or cell lysate to the sensor surface causes a signal that masks the signal from the analyte of interest (Figure 5). This becomes detrimental for serum components like IgE or cytokines that are present in low concentrations. However, the study of Battaglia et al. demonstrated the detection of biologically relevant levels of the cytokine IL6 in cell culture media using a SPR sensor. For reducing the non-specific protein adsorption, the sensor surface was modified by a layer of NHS ester and 16-mercaptohexadecanoic . Weinhart et al. suggested SAMs of linear polyglycerol derivates for gold surfaces. The study that focused on the protein adsorption to modified gold surfaces revealed that linear polyglycerol can be alternatively used for PEG as a protein resistant coating material .
Compared to microarrays and bead assays, label-free methods have the key advantage that additive detection antibodies or fluorescently labeled samples are not needed. Fluorescence detection requires the labeling of antibodies which is cost-intensive and may influence the bioactivity of the target molecules. The use of fluorescent dyes, either in patient samples or conjugated to detection antibodies, requires adapting the optimal S/N ratio to ensure a sensitive analysis with low background noise. Furthermore, an inefficient sample labeling interferes with the interaction of the target molecule and consequently leads to a loss of sensitivity. Another bottleneck is that the application of multiplex immunoassays assumes the use of high affinity antibodies, i.e., approximately 1011–1012 M−1. All antibodies used in an assay should have closely comparable affinity constants to ensure the sensitivity of the test .
Nowadays, the awareness of the complexity of miscellaneous diseases is continuously increasing. Infection- or autoimmune diseases are influenced by various factors. Not an individual marker, but rather a panel of well-characterized marker molecules, defines the clinical picture, allowing a stratification of patients. When utilized for prognostics, the amount of molecules increases exponentially in order to study different disease states. Living up to the expectations of modern medicine makes the necessity of multi-parametric assays apparent. Multi-analyte immunoassays have the potential to be employed as the state-of-the-art technology for the in-depth serum biomarker discovery including validation studies and assay development. The performance strength of each immunoassay platform should be considered allowing their appropriate application in the process of biomarker discovery. Microarrays have the capacity for an unbiased and rapid serum screening to identify novel protein biomarkers from serum . The development of a sensitive and specific microarray platform requires the choice of an appropriate surface chemistry, as well as the quantification of signal intensities, and the S/N ratio. To ensure the diagnostic significance of a protein biomarker a validation process is required. This profound validation process of the preselected biomarkers comprises an analysis concerning their specificity and sensitivity, and the definition of the assay conditions. Array-based SPR systems are exceedingly suited for this purpose since they provide kinetic data and allow a relatively high-throughput. The kinetics of the preselected interactions are analyzed by performing an affinity ranking and the molecules with the favored characteristics can be chosen for diagnostic assay. The validated molecules and the defined assay conditions are implemented in a laboratory friendly bead assay to reconsider the clinical utility. Biomarker candidates, that are inappropriate for clinical diagnostics can be excluded early on, before moving with that candidate in clinical trial.
The authors would like to thank Michal Or-Guil and colleagues for their assistance with peptide arrays, Nina Babel and colleagues for their support concerning clinical issues, Robert Wellhausen and Sarah Schumacher for helpul discussion. This work was supported by the European Regional Development Fund.
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|Surface coating||Immobilization mechanism||Chemical bounding||Advantages/Disadvatages|
|Nitrocellulose||Hydrophobic adsorption||Non-covalent||No requirement for addit. coupling reagents nor antigen modification/weak binding, loss of activity, high S/N ratios|
|Poly-Lysine||Electrostatic Forces on charged surface||Non-covalent|
|NHS ester||Lysine residues react with active ester to form amid bounds||Covalent||Stable interaction/unstable in aqueous solution, not orientated|
|Aldehyde||Primary amino-groups react with the aldehyde surface||Covalent||Stable interaction/not orientated|
|Epoxy||Nucleophilic residues (NH, SH) react with epoxy||Covalent||Stable to hydrolysis at neutral pH/not orientated|
|Streptavidin||Biotinylated residues bind to streptavidin||Strongest non-covalent bounding in nature 10−14 M||Site-specific immobilization, strong interaction/antigen modification|
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

domain: chemistry


text:
Q: Describe what you publish in 25 characters or less.
A: non-linear multi-threaded
Q: What other current publications (or publishers) do you admire most?
A: I admire Tor for publishing stuff I've read for years, I admire anyone publishing in Twine (both narratives and games), I love Inkling and Comixology for pushing the boundaries of what an ereader can do. I also love every tiny little press in the world, because they are all run by people who are passionate about words over money.
Q: If you publish writing, who are your favorite writers? If you publish art, who are your favorite artists?
A: That's like asking my favorite food: it changes all the time. This minute, it's Helen Macdonald, Carlos Ruiz Zafon, Roberto Bolaño. Next week, it'll be someone else.
Q: What sets your publication apart from others that publish similar material?
A: Right now, we're the only publishers using Lithomobilus, an ereading platform that makes multi-threaded work possible. We're also all about community - our writers are our friends and we encourage them to be each other's support systems and cheerleaders.
Q: What is the best advice you can give people who are considering submitting work to your publication?
A: Don't email any of the editors, and for heaven's sake, read the guidelines. I am serious about rejecting out of hand anything that comes in single spaced. I'm too old to want to spend any time squinting at my screen.
Q: Describe the ideal submission.
A: The ideal submission has a cover letter that includes a very short (50-60 word) bio, and very little else. The manuscript is in Times New Roman, double spaced. The piece's relationship with the theme of the issue for which it's being submitted is obvious and clever (it's not just a re-telling of the story, it's not related only tenuously by some vague theme or feeling). If it's nonfiction or nonfiction, the narrative is compelling, the language is clear and precise, the author's use of imagery is apt. If it's poetry, the language is lyrical and beautiful.
That said, I am not the only person reading for Zoetic Press. I am regularly overruled by the rest of the reading staff who like different things than I like.
Q: What do submitters most often get wrong about your submissions process?
A: The thing most often gotten wrong is that people email me personally. While I'm happy to answer questions about Lithomobilus or about what kinds of things Zoetic Press publishes, I am not going to read your pitch via email. If you want to submit your novel for consideration, our submission guidelines are right there on our website where authors can find them and submit via our Submittable page.
Q: How much do you want to know about the person submitting to you?
A: I am not impressed by previous publications or fancy degrees. We've published both first-time authors and award-winning authors in our pages. In fact, I normally don't even read the cover letter until after I've made up my mind about the story itself.
Frankly, I'm a little put off by anyone who seems like they're trying to impress me with their list of authoring credits (we get it, you've published everywhere, that's great) or worse, by name-dropping other authors. There's a difference between "my friend John Smith was published in your first issue and told me my material might be right for you" and "Joan Didion said that my first book was the best thing she's ever read." If your friend is recommending us to you, that's great, and I'm excited. But if you're telling me that someone else is recommending you to to me, I'm capable of making up my own mind.
Q: If you publish writing, how much of a piece do you read before making the decision to reject it?
A: I generally give something 3 or 4 pages. My standard test is this: if, in the first three or four pages, I'm overcome with the urge to check my Facebook page while reading a story, I'll give it a pass. If I can't read it in one sitting without feeling like I need to take a break and do something else, I can't expect my readers to do any differently.
Q: What additional evaluations, if any, does a piece go through before it is accepted?
A: Pieces are read and voted on by at least 4 different readers before they're accepted. If we're on the fence about a piece, there's often a discussion where the people who are passionate about it have to advocate for it. There are some pieces about which we're unanimous, and those are the easiest, but if we're not, factors like how well a thing makes use of our unique platform, what audience it's written for, etc. come into play.
Q: What is a day in the life of an editor like for you?
A: When I'm being Editor me (as opposed to any one of the million other things I do), it looks like having a meeting with my managing editor where we talk about what we're putting in the blog this week, what we're putting in our weekly editor's letter, what projects on in the works and what stage everything's at. I read some stuff and vote on it. Once a month or so, the managing editor and I get together and go over all the things that have been voted on and choose what gets published. And every other month, I get to do that thing where I sit down and cut checks to all the writers for our publications. I have to be honest, that's one of the most fun parts for me.
Q: How important do you feel it is for publishers to embrace modern technologies?
A: There are certain parts of technology, like electronic submissions, that I feel every publisher should make use of. Any publisher who doesn't accept electronic submissions is risking having their stream of submissions dry up as authors consider other presses that don't require the time and expense of paper mail. Online social networking is great for building a community around your press and your authors, but I don't know that it's crucial to success. I think that there is still room for boutique paper printing for projects that can only be done in paper. That said, I think that the possibilities of digital are only just now starting to be revealed, and that people are far more exited by them than by anything being done with paper.

domain: literature


text:
Love actually, has such an ill fated reputation. It’s proceeded by new age tinder hook ups, expressions of sexual freedom, the quick sand of never lasting. The endless possibilities of greener pastures winking at us from a distance. A world in which the shallow excitement of lust lingers in the air like fragrance.
Then there’s another world, where souls skilfully take charge of their very own destiny. Challenging limitless opportunity, all within their own inner realms. No real need to be seduced beyond themselves. By all means, I am not an old romantic, I didn’t exactly buy into all the love band aid melodrama. I in fact, believed in loving everyone unconditionally without limiting labels. However, whilst floating through this world of seeker souls, I literally fell in the mystical clutches of the cosmic love guru. Not at all wanting a lover of any kind.
He wasn’t teasing me with the idea of love. Hell no. He was offering unconditional love on a delicious platter of all those endearing qualities I ever wanted to find in others. All of it, in one single soul. Hard to believe as it is, one late evening, I found myself window shopping on a website for lonely souls. I was lonely less, bored more. At the time, something told me to stay online. Within moments, my senses tingled as the words on this page became electric. They were alive, singing in an intoxicating chant. My fingers unable to resist, with a will of there own, charging masterfully across my keyboard. Typing a sentence. Just one sentence.
“Cosmic calling. How do you reply?”
Sighing in relief, I inhaled. It was as I if had suddenly been compelled to reply. Almost as if I had no choice. It was pure madness. He lived in the US. He wasn’t even looking for anything. Actually, I wasn’t looking for anything. He would never reply. With that thought in my mind, I shut down my laptop. Tonight, I was being stranger than my normal strange. Switching off the lights, I tucked myself away in my bed.
“He will reply.” Whispered my heart.
I fell into a sleep listless. Dreaming of more peculiarity than the norm. It was the 1st of July 2016. This date is imprinted on my soul. I will never forget it, regardless of the outcome. The energy charging my heart was incredibly powerful. I would never forget that feeling.
It wouldn’t be a love story, if he hadn’t replied. But we have a love story, so the cosmic love guru peddled his magic, wielding a wonderous path for us to travel. I feel in love with him, instantly. I think I had no choice. It was as if Cinderella’s fairy godmother had sprinkled magic dust and my Prince Charming, just stopped by whilst searching for the owner of one glass slipper.
His soul is beautifully honest, filled with love and laughter. He is like the rays of sunshine. He is the moonlight, brightening up midnight pathways. The fact that we live in two different countries, was really no problem at all. Nothing was an obstacle within our unfolding love story. The time difference providing us with creative opportunities to express our gratitude for one another. Honestly, this was an uncomplicated long distance “platonic friendship- romance”. I believe that boils down to the mere truth- that we were individually fulfilled souls, working on our vulnerabilities. We were not willing to check into drama cycles craving to be rehashed.
I am sure the cosmic love guru stood proudly, when for the first time we locked eyes in Barbados 6 months later. Our souls finally meeting in the flesh. We were not strangers to one another. Our physical bodies, only completing our hearts messages. We had met long, long before. We needed no one to tell us otherwise. Three months later, in April 2017, we forged our alliance, in the way of a cosmic marriage. Together, we returned to our one home, our hearts. Now, this is where we live. In each other’s hearts, bursting with this explosive, passionate, crazy self consuming love. Our souls are on fire. Burning in the wander of our magical union.
I wish we could meet the cosmic love guru, to thank him for his blessing grateful. We will forever cherish this gift.
Eternally grateful souls. We look forward to our future as one.
Stardust drifting in the wings of the wind.


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text:
‘Internal review’ launched amid reports of COVID test hiccup
The Ministry of Health will be launching a “internal review” following reports that a person who tested positive for COVID-19 was given a ‘clearance letter’ that indicated a negative test result.
Reports are that this COVID patient is the person who attended a funeral at the Sea Cow’s Bay Methodist Church last Saturday, March 27.
And in a news article published on Tuesday, 284 Media reported that government officials “allowed the public to believe that the individual breached quarantine and deliberately put others at risk”.
Health Minister Carvin Malone described the article as “unfortunate”, considering that officials are still investigating what happened.
“The facts have to be retained before they can reach that type of conclusion,” Malone told BVI News.
Nearly 30 thousand successful procedures
The minister further said he is confident in the territory’s laboratory facilities which have conducted nearly 30,000 successful tests since being established.
Notwithstanding this confidence, the minister said “the [recent] activities warrant an internal review”.
“And if it is determined that any procedures were not followed, it will then give us the ability to correct it and continue on to our thirtieth thousand swab,” he told our news centre.
While further touting the work of the territory’s COVID lab, Malone added: “I think we have a very good record and we’ll actually defend it. But there is nothing really to defend. We just have to do the review and see if there is anything that we need to do better.”
Health Ministry contact tracing as funeral home temporarily closes
Copyright 2023 BVI News, Media Expressions Limited. All Rights Reserved. This material may not be published, broadcast, rewritten or distributed.
THEM NEED TO ANSWER THE DOGGONE PHONE
Here we go again blaming somebody else
Are you going to explain how the Filipino lady died from Covid 19 last year after several calls to the hospital for help?
Don’t take her husband’s word for it. There is more to that story.
It’s a simple thing really. Type Y for positive, N for negative.
Clear the family name and let them mourn in peace. Meanwhile, get your apology statement in order as your department placed so much stress on individuals and family who attended.
Politician with a Scottish name.
This is what happens when people lie too much! They let days pass with the person being blamed for this and come at the last hour trying to save face talking about investigation. We are still awaiting the investigation findingd into the lady that died last year and theh listed it as our omly covid death. Where are the findings? So sick of the f**king nonsense in this place!
Sad Situation! Height of Negligence! All involved from the Individual who answered the Phone to the Physician who discharged her in the First Incident(to include Emergency and Health Personnel) should be held accountable. “To cause no harm” surely was not the Protocol here.
Somebody needs to look into the recent change in the BVIHSA laboratory management. It is in shambles!
Maybe they need to hire qualified people.
The credibility and transparency of this administration needs and investigation. Simply put Funeral was Saturday today Tuesday before they could make a public address on something of this magnitude. This so called error if it was like other countries could have cost lives but realistically we knew something smelt fishy. While others were hastily jumping to conclusions the present administration continues to spread misinformation about people who are vaccinated doesn’t need to quarantine. That is both misleading and dangerous and there is no such literature stating this anywhere unless he has a special AstraZeneca hand book. CANADA recently paused along with Germany concerning serious adverse reactions. I need not say anymore, ill sit back and continue to let them shoot themselves in the foot.
By the way as educated people we need to also be more informed as to what’s going on in the world. The silent war that’s happening, economic attacks which trickles down 5o everyone’s livelihood.
They are always quick to put the blame on the victim.
I remember the M……r himself blaming the Phillipino lady when she got sick only to find out later that the lady had gone to the hospital to get help and they misdiagnosed her and sent her home
And the person that died last year? Investigating that or still ignoring it since it was “only” an expat?
How did they find out the person is positive after giving him a negative report????
and the pieces are falling into place \ where are all the experts finger pointers ?
Enough. Hopefully we don’t have to wait 23 months to go to the polls

domain: news


text:
We have used Wordpress for the website [login to view URL] which was launched five years ago. Following a recent Wordpress update the top menu does not appear anymore. We need someone experienced with Wordpress to help update the template to ensure the top menu loads properly.
When you first open the website you may notice the top menu shows up for 1 second then disappear.
Another change that is needed is to add a News section and to have the three latest news/blog posts appear on the home page in the section currently occupied by the newsletter box and the Facebook plug in.
We want to add the logo in the footer at the left, and replace the Contact Us with the Facebook plugin that was on the section above.
Finally, on the Support and Contact Us pages, we need to add Captcha before the Submit button to reduce the amount of spam.
Budget for this project is CAD100 and shouldn't take long for someone experienced with Wordpress.

domain: design


Sequential Information Elicitation in Multi-Agent Systems
Rann Smorodinsky, Moshe Tennenholtz
We introduce the study of sequential infor-mation elicitation in strategic multi-agent systems. In an information elicitation setup a center attempts to compute the value of a function based on private information (a-k-a secrets) accessible to a set of agents. We consider the classical multi-party computa-tion setup where each agent is interested in knowing the result of the function. However, in our setting each agent is strategic,and since acquiring information is costly, an agent may be tempted not spending the efforts of obtaining the information, free-riding on other agents' computations. A mechanism which elicits agents' secrets and performs the desired computation defines a game. A mech-anism is 'appropriate' if there exists an equi-librium in which it is able to elicit (sufficiently many) agents' secrets and perform the computation, for all possible secret vectors.We characterize a general efficient procedure for determining an appropriate mechanism, if such mechanism exists. Moreover, we also address the existence problem, providing a polynomial algorithm for verifying the existence of an appropriate mechanism.
PDF Link: /papers/04/p528-smorodinsky.pdf
AUTHOR = "Rann Smorodinsky
and Moshe Tennenholtz",
TITLE = "Sequential Information Elicitation in Multi-Agent Systems",
BOOKTITLE = "Proceedings of the Twentieth Conference Annual Conference on Uncertainty in Artificial Intelligence (UAI-04)",
PUBLISHER = "AUAI Press",
ADDRESS = "Arlington, Virginia",
YEAR = "2004",
PAGES = "528--535"


text:
More than 60 health care professionals from the region attended the first-ever Treating Addiction: Integrating Medication-assisted Treatment (MAT) into Primary Care conference on Jan. 25 in Auburn.
Western Sierra Medical Clinic organized the half-day conference to help health care professionals combat substance abuse in our community, especially for residents addicted to opioids such as prescription pain medications and heroin, a critical and fast-growing problem in the region.
Western Sierra has five podcasts that feature the speakers and their valuable information shared at the conference.
As you will hear, collaboration is key to addressing and defeating the problem. We must continue to develop new partnerships, explore new opportunities and improve existing MAT services. The conference is just the latest of a community wide effort to help those battling substance abuse – and an excellent example of Better Health Together.
*Click on the image to the left for a PowerPoint of the Presentations*
If you have any questions or would like more information about Western Sierra’s MAT program, the conference or the featured speakers, please contact Cheryl Rubin at firstname.lastname@example.org.
Thanks to the Center for Care Innovations for its partnership and support for this conference.
- Scott McFarland, CEO, Western Sierra Medical Clinic
- Brian Dahle, Assembly Republican Leader, District 1
- Christina Lasich, MD, Pain & Addiction Medicine and Chief Medical Officer, Western Sierra Medical Clinic
- Alinea Stevens, MD, MPH, Chapa-De Indian Health
- Veronica Velasquez-Morfin, MD, Medical Director, El Dorado Community Health Centers
- Rob Oldham, MD, Placer County Public Health Officer & Western Sierra Medical Clinic Board member
- Michael Freeman, Manager, Substance Abuse Disorder Compliance Division, CA Dept. of Health Care Services
- Ramiro Zuniga, MD, Medical Director, California Health & Wellness
- Mike Witte, MD, Chief Medical Officer, California Primary Care Association
- Sarah Khawaja, CA Hub and Spoke Grant Manager, Aegis Treatment Centers

domain: medical


text:
Strausbaugh/DeLay:Information about Fanny Fay
Home Page |Surname List |Index of Individuals | |Sources
Fanny Fay (b. Abt. 1801, d. date unknown)Fanny Fay was born Abt. 1801 in North Carolina, and died date unknown in Bollinger, Missouri.She married Samuel Virgin, son of William Virgin and Sophia Unknown.
Children of Fanny Fay and Samuel Virgin are:
- +Enoch Virgin, b. April 22, 1830, Bollinger, Missouri, d. December 18, 1905.

domain: history


Neha Kini heads the IT division at Vedanta-Sterlite Copper. She has been with the company for 17+ years and has an expertise in strategizing, planning and executing IT-led business transformation projects. Kini is also a former CIO100 honoree.
IT Change management is crucial for any organization as every IT landscape undergoes change in one or the other way. As more and more business processes are being automated, a failure to manage change well will result in a failure to meet service commitments.
Vedanta-Sterlite Copper implemented a one-stop solution manager to automate end-to-end process, manage the incidents, changes and problems centrally using the defined change management and incident management process. The multiple standard roles in SOLMAN tool also facilitates proper authorization with segregation of duties. Also there is efficient and smooth project management. Real time status reporting and tickets processing with dashboards is available.
The benefits from the project were multifold for the organization. There were improved IT initiatives management, and release management with better user experience. IT key user wise ticket distribution was enabled for monitoring the key user efficiency. It also helped to improve compliance requirements and audit ratings. It maps to all functions and has a simple but flexible user interface. Lastly, the solution manager provides detailed reporting and monitoring on system health and performance thereby confirming system availability.


text:
Tangerine Dream Color Palette Printable Wall Art
Unleash Inspiration with Customizable Color Palette Wall Art!
Elevate your space with vibrant and unique décor inspired by your favorite color palettes from Causeways Palette on Pinterest.
Tangerine Dream is a colorful palette or red, orange, warm yellow, a light sage green and a purple! Perfect to brighten up any space!
What You'll Receive:
Upon purchase, you'll instantly download a zip file containing high-resolution PDFs meticulously crafted to offer versatility in framing options. Each color palette design is available in six ratios, ensuring compatibility with various frame dimensions.
- 2:3 Ratio
- 3:4 Ratio
- 4:5 Ratio
- 5:7 Ratio
- 11:14 Ratio
- 12:12 Ratio
Included with your download are comprehensive instructions guiding you through the printing process. These guidelines ensure optimal print quality, suggesting paper type, settings, and framing suggestions for each ratio.
Personalize Your Space:
Create a personalized gallery wall or accentuate any room with these customizable color palette artworks. Express your unique style by mixing and matching ratios or frame them individually for a captivating visual impact.
Dive into your creativity and infuse your living space with the essence of your favorite color schemes. Transform your walls into a canvas of inspiration with our versatile, printable wall art!


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Queen's bank Coutts warns thousands of investors on risky deals
Coutts, the Queen’s bank, has written to thousands of its customers to warn them they may have been mis-sold risky investments.
The City watchdog the Financial Conduct Authority has ordered the 322-year-old private bank to contact all its UK investors, amid concerns that it has been handing out unsuitable advice for decades.
It said up to 15,000 clients, who typically have a minimum of £1million to invest, will be contacted. The first letters were sent out last month. In the letter, chief executive Michael Morley said: ‘Looking back, there have been some instances where the advice given during our previous advice process could have been better, and we are working hard to address that.’
He adds that customers who ‘have suffered any financial detriment’ will be ‘compensated in full’.
This is just the latest black mark against Coutts, which is owned by state-backed Royal Bank of Scotland and has a cash machine in Buckingham Palace. It was fined £6.3million by regulators in 2011 for mis-selling investments backed by failed US insurance giant AIG.
Comedian Frank Skinner and Sir Keith Mills, founder of Air Miles and Nectar cards, lost millions of pounds in the fund, which was billed as a low risk alternative to deposit accounts. They launched high profile campaigns to win their money back.
It was also fined £8.75million in 2012 for ‘serious and systematic’ failings when handling money from suspected criminals or foreign despots.
The latest mis-selling scandal relates to more general concerns from regulators that Coutts sold unsuitable high risk investments to savers.
The FCA is also concerned Coutts failed to keep proper records of its advice, making it impossible to prove it was suitable. The firm will review advice given to all customers who took out investments before November 26, 2012 when it improved its advice processes.
Most watched Money videos
- Ray Massey gets exclusive access to the new green Rolls-Royce Spectre
- How will the looming recession affect YOU?
- 1990's advert for the Vauxhall Cavalier Turbo 4x4
- Rustbucket Mercedes-Benz roadster sells at auction for almost £40k
- Ford tests traffic light tech that could change the commute forever
- Bentley brings the comfort of a limo to their popular SUV
- Watch the BANNED Land Rover ad that 'misrepresented' parking sensors
- Ford unveils electric version of Britain's best-selling vehicle
- A rare 1955 Mercedes 300 SLR Coupe being driven at Goodwood
- RM Sotheby's has sold a Mercedes for a record £115million
- Renault previews new family car powered by a hydrogen-electric system
- Land Rover unveils its new £80,000 Range Rover Sport


text:
05 Feb Jollibee, To Open First Ever Branch in Balingasag Soon
Another one of the country’s largest fast-food chains reveals plan to open a branch in Misamis Oriental soon.
With the recent news of a Mcdonald’s branch in El Salvador, an upcoming Jollibee branch was also announced with a job hiring post in Balingasag, Misamis, Oriental.
According to Aljun Acenas Fermo’s post, Jollibee is currently looking for a Management Trainee for the Balingasag branch with Bachelor’s Degree in Food or Business related courses.
Interested applicants should send their application to email@example.com or visit their Human Resource Office at Basoy Bldg. 1, Brgy. 4 National Highway, Balingasag, MisOr and look for Ms. Annie Torentira.
You can also inquire with these number mga higala: 0917-7170-443
Source: Aljun Acenas Fermo


What kinds of relays can be divided according to the principle of action?
1. Electromagnetic relay
A relay that produces a predetermined response by the relative motion of mechanical components under the action of current in the input circuit.
It includes DC electromagnetic relay, AC electromagnetic relay, magnetic holding relay, polarization relay, reed relay, energy-saving power relay.
(1) DC electromagnetic relay: The electromagnetic relay in which the control current in the input circuit is DC.
(2) AC electromagnetic relay: The electromagnetic current relay whose control current in the input circuit is AC.
(3) Magnetic holding relay: The magnetic steel is introduced into the magnetic circuit. After the relay coil is de-energized, the armature of the relay can still maintain the state when the coil is energized, and has two stable states.
(4) Polarized relay: A DC relay whose state changes depending on the polarity of the input excitation amount.
(5) Reed relay: A relay that opens, closes, or switches a line by a action of a reed that is sealed in the tube and has a dual function of a contact reed and an armature magnetic circuit.
(6) Energy-saving power relay: The control current in the input circuit is an AC electromagnetic relay, but its current is large (generally 30-100A), small in size, and power-saving function.
2. Solid state relay
A relay that performs input and output functions by electronic components without mechanical moving parts.
3. Time relay
When the input signal is added or removed, the output portion needs to be delayed or time-limited until the specified time to close or open the relay of its controlled line.
4. Temperature relay
A relay that operates when the outside temperature reaches a specified value.
5. Wind speed relay
When the speed of the wind reaches a certain value, the controlled circuit will be turned on or off.
6. Acceleration relay
When the acceleration of the moving object reaches the specified value, the controlled circuit will be turned on or off.
7. Other types of relays
Such as optical relays, acoustic relays, thermal relays, etc.


2004 dodge neon
Hey so I have a 2004 dodge neon Does need some body work Car has been lowered Rear tint Comes with two sets of tires Ran very well during the winter. Always started. New clutch done right ...
1998 Dodge Neon Sedan
car runs fine has a very slow oil leak and an even smaller resivoire leak on the break like.
2001 Dodge Neon Coupe
2001 Dodge Neon Has 4 newer tires and wheel bearings within the last 6 months. Engine needs work - not sure what is wrong with it. The car is located in Nanton so it will need to be towed. ...
2000 Dodge Neon Sedan
very clean one owner well maintained new tires brakes,inspection with sale no body damage good glass needs nothing just drive away.


text:
* Some 1,500 gather outside parliament
* Protest over possible changes to electoral boundaries
* Government wants top court to rule on the issue
By Sylvia Westall
KUWAIT, Sept 10 (Reuters) - Around 1,500 Kuwaitis took part in a rally late on Monday to protest against possible changes in the electoral law and call for more democracy.
The crowd, which included opposition lawmakers and political activists, gathered outside parliament in a square which has hosted several anti-government demonstrations since late last year.
Monday’s turnout was smaller than previous rallies in the major oil producer and there was only a light police presence.
Kuwait’s Interior Ministry said on Sunday it would act firmly against any “unlicensed” protests in the country and said calls for a sit-in at Erada Square undermined security and threatened public order.
While the U.S ally and OPEC member state has not had the kind of mass popular uprisings seen elsewhere in the Arab region, political tensions have escalated between the hand-picked government and elected parliament.
“Our problem is with the government. We like the emir, we all do,” Suleman Mohammed, who is retired from the army, said.
“We want a little more democracy, more freedom, more rights.”
While Kuwait has one of the most open political systems in the Gulf, the head of the ruling family, Emir Sheikh Sabah al-Ahmad Al-Sabah, has the final say in political matters.
He chooses the prime minister who in turn picks a cabinet, with important positions held by al-Sabah family members.
It is a government appeal to Kuwait’s top court which is at the heart of the latest political tension.
The cabinet has asked the constitutional court to rule on the 2006 electoral law which divides the country into five constituencies. It says the verdict is needed to protect the outcome of future elections from possible legal challenges.
But some opposition figures say this is a bid to abolish the current boundaries and gerrymander victory in elections expected this year or next. The court is due to rule on the government’s petition later this month.
The court drew the anger of protesters earlier this year when it effectively dissolved the opposition-dominated 2012 parliament and reinstated a more government-friendly assembly.
“The government plays with our democratic system. There are people behind the government, controlling it,” said a 33-year-old government ministry employee who gave his name as Abu Saad.
He said he was referring to divisions within the ruling family and competition between other powerful families.
“Kuwait is like a cake and everyone wants a piece,” he added. “Twenty years ago Kuwait was number one in the region, there was no Dubai, no Qatar. Now it is in last place.”
Years of political infighting have stalled investment in Kuwait, which is one of the richest countries in the world per capita thanks to its oil wealth and small population.
Speakers at the rally, who included Islamists and tribal members of parliament, renewed opposition calls for an elected government. Political parties are banned in Kuwait so lawmakers form blocs in parliament based around tribal ties and policy.
Turnout at the height of protests last year over political corruption peaked at tens of thousands and during one rally protesters stormed parliament. (Editing by Alison Williams)

domain: sociology


text:
The Japanese Bankers Association is considering a review of as many as 16 banks to confirm that they are following the lobbying group’s guidelines for submitting Tokyo interbank offered rates.
As many as 15 reference banks may be reviewed for how they submit their euro-yen Tibor rates, said Hisanao Aoki, a spokesman for the association.
Barclays PLC last month was fined ￡290 million ($453 million) for rigging London interbank rates, prompting authorities in Europe, Asia and the U.S. to broaden inquiries into manipulation of benchmark gauges for borrowing costs. Citigroup Inc. and UBS AG in December were ordered by Japanese regulators to suspend some operations after the banks’ staff were found to have attempted to influence Tibor rates.
“There’s growing pressure from Japanese lawmakers on the banking industry to determine and confirm that Tibor has been properly submitted and set without any wrongdoing,” said Shinichi Nakamura, an analyst at SMBC Nikko Securities Inc. “If any bank is found to have attempted manipulation of the rate, it would be a huge problem.”
The Democratic Party of Japan plans Thursday to call on the banking lobby to explain how Tibor is set, lawmaker Tsutomu Okubo said Tuesday. The former Morgan Stanley banker is heading a panel examining financial firms.
The reference banks are responsible for submitting interbank offered rates to the association, which compiles and sets the benchmark lending rate, according to the lobby’s website.
Monex eyes Sony broker
Online securities firm Monex Group Inc. will acquire and turn Sony Bank Securities Inc., a wholly owned online brokerage arm of Sony Bank, into a wholly owned unit and merge it with Monex Inc. this business year.
The planned acquisition, scheduled for Aug. 1, is aimed at broadening Monex’s customer base.

domain: finance


text:
Thank you for helping us by reporting bad content.
Hey There Everyone!
Jessica Stroup attended the Badgley Mischka Fall 2009 show during New York Fashion Week, wearing a white sweet-heart necklined modified version the Pre-Fall 2009 gown, with a feathered skirt, and jeweled bodice. LOVE THE DRESS! It`s adorable, super cute! Eva Longoria, also opted for a modified version, when she arrived at the premiere of The Perfect Game during the Guadalajara International Film Festival in Mexico. I prefer the shorter version of Jessica so she wins for me :)).
Who Wore Badgley Mischka Better?
- Jessica Stroup
- Eva Longoria
*images do NOT belong to me, please see source link below for more information*

domain: celebrity


text:
Brazilian Stock Decline Led by Itau as Valuations Seen Priceyby
Lender contributes most to decline after three-day rally
Investor optimism gives way to caution on political outlook
Brazil’s Ibovespa dropped for the first time in three days as lender Itau Unibanco Holding SA tumbled after reaching its highest valuation in three weeks.
Itau was the biggest drag on the benchmark equity index as it traded at 9.9 times estimated earnings, the most expensive since Sept. 8. Financial stocks extended losses, joining a global rout, after some funds that clear derivatives trades with Deutsche Bank AG were said to have withdrawn excess cash and positions held at the German lender in a sign they lack confidence. The depreciation of the real buoyed pulp producers Fibria Celulose SA and Suzano Papel e Celulose SA, which get most of their revenue from exports.
The Ibovespa has advanced 35 percent this year on optimism that a new government will be able to tame a political crisis and eventually pull Brazil out of its worst recession in a century. In the past six weeks, however, investors have adopted a more cautious approach to the country’s stocks as they assess the pace of the recovery and prospects for commodity producers. Average trading volume of stocks this quarter is 10 percent below that of the three months ended in June, according to data compiled by the exchange.
"There’s so much anxiety over the turnaround plan and the political scenario that the market has hit the break, waiting for more news to support allocation decisions," Paulo Henrique Amantea, an analyst at the brokerage Guide Investimentos, said from Belo Horizonte, Brazil.
The Ibovespa slumped 1.7 percent to 58,350.57 at the close of trading in Sao Paulo. Itau lost 2.5 percent as Banco Santander Brasil SA retreated 2.7 percent. Fibria, which also announced a price increase for clients in Asia, added 2 percent and Suzano rose 2.5 percent. The real weakened 1.3 percent.


text: Thousands of physicians worldwide have cured millions of covid patients in a matter of days without side effects, using effective and safe treatments. All these cures are backed by scientific studies that confirm their efficacy. There are for example ivermectin, HCQ + zinc, quercetin + zinc, budesonide, CDS, and nebulized hydrogen peroxide. Perhaps the greatest crime against humanity, during this planned pandemic, is that these cures are suppressed by governments, Big Tech, health organizations and news media, in order to further the agenda of imposing gene altering, nanotechnology injecting experimental vaccines onto the entire world. Nobody ever needed to die from covid. Every death is the direct result of the criminal suppression of the available treatments, and the murderers will be held responsible for their crimes against humanity.