text:
Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.
23 Related JoVE Articles!
Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Institutions: University of Arkansas for Medical Sciences .
Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3
. As a result, these enzymes are prime targets for small molecule studies and drug development 4
. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5
. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5
Molecular Biology, Issue 61, LSD1, lysine demethylase, mass spectrometry, reductive methylation, demethylase quantification
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2
used the ab initio
accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio
folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5
. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
Measuring Peptide Translocation into Large Unilamellar Vesicles
Institutions: Wellesley College, .
There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1
. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3
. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5
, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7
. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8
. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13
. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15
. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17
. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18
, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.
Molecular Biology, Issue 59, membrane translocation, vesicle, FRET, peptide, tryptophan
Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Institutions: Fred Hutchinson Cancer Research Center - FHCRC, University of Victoria, Broad Institute of MIT and Harvard, University of Victoria, Plasma Proteome Institute.
There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1
An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA
(Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2
In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS
) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4
and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7
To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID
) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13
In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.
Molecular Biology, Issue 53, Mass spectrometry, targeted assay, peptide, MRM, SISCAPA, protein quantitation
Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery
Institutions: The Methodist Hospital Research Institute, National Center for Nanoscience and Technology.
The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3
The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5
Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6
Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9
Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples.
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11
Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.
Bioengineering, Issue 62, Nanoporous silica chip, Low molecular weight proteomics, Peptidomics, MALDI-TOF mass spectrometry, early diagnostics, proteomics
Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation
Institutions: Tunghai University, University of California, Riverside .
Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3
. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4
. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2
, instead of a HPLC/ESI (electrospray ionization)-MS system5,6
. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2
). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9
Biochemistry, Issue 57, Deuterium, H/D exchange, Mass Spectrometry, Pak2, Caspase 3, MALDI-TOF
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
Institutions: China Medical University , University of Arkansas for Medical Sciences , University of Arkansas for Medical Sciences .
A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1
). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1
. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2
). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2
in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105
cells per well typically used in ELISPOT assays1,3
, 1,000 T-cell clone cells in the presence of 1 x 105
autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4
Immunology, Issue 61, Interferon-γ enzyme-linked immunospot assay, T-cell, epitope, human papillomavirus
Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus
Institutions: Macalester College.
Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen, or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.
Neuroscience, Issue 82, Natural Science Disciplines, Life Sciences (General), Behavioral Sciences, mechanical hyperalgesia, mice, electronic pressure meter, inflammation, snake venom
Determination of the Gas-phase Acidities of Oligopeptides
Institutions: University of the Pacific.
Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6
. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8
, the results are often complicated by solvent effects 7
. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10
. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3
CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1
. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2
). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.
Chemistry, Issue 76, Biochemistry, Molecular Biology, Oligopeptide, gas-phase acidity, kinetic method, collision-induced dissociation, triple-quadrupole mass spectrometry, oligopeptides, peptides, mass spectrometry, MS
A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Institutions: Dartmouth College, University of Rhode Island, Dartmouth College.
Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2
. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5
. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.
Biochemistry, Issue 85, Immunoassay, Protein Immunogenicity, MHC II, T cell epitope, High Throughput Screen, Deimmunization, Vaccine Design
Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)
Institutions: Louisiana State University Agricultural Center.
We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al.
2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus
(Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al.
1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al.
(2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii
, Holomastigotoides hartmanni
), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro
(protozoa culture) and in vivo
(microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.
Microbiology, Issue 46, Isoptera, Coptotermes formosanus, Formosan subterranean termite, termite control, paratransgenesis, symbionts, anaerobic, fluorescence
Identifying Protein-protein Interaction Sites Using Peptide Arrays
Institutions: The Hebrew University of Jerusalem.
Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.
Molecular Biology, Issue 93, peptides, peptide arrays, protein-protein interactions, binding sites, peptide synthesis, micro-arrays
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides
Institutions: The Hebrew University of Jerusalem.
In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro
will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2
. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8
. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a "beads on a string"-like structure by this process.9
The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.
Chemistry, Issue 81, Materials (General), self-assembly, peptides, diphenylalanine, atomatic interactions, coassembly, molecular recognition
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Institutions: University of Heidelberg.
All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1
H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g.
crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.
Chemistry, Issue 81, Molecular Chaperones, mass spectrometers, Amino Acids, Peptides, Proteins, Enzymes, Coenzymes, Protein dynamics, conformational changes, allostery, protein folding, secondary structure, mass spectrometry
Peptide-based Identification of Functional Motifs and their Binding Partners
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Institutions: Stuttgart University.
Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.
Biochemistry, Issue 93, Peptide arrays, solid phase peptide synthesis, SPOT synthesis, protein lysine methyltransferases, substrate specificity profile analysis, lysine methylation
Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library
Institutions: The Scripps Research Institute.
Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1
) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.
Chemistry, Issue 88, Split-and-pool synthesis, peptide tertiary amide, PTA, peptoid, high-throughput screening, combinatorial library, solid phase, triphosgene (BTC), one-bead one-compound, OBOC
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
A Protocol for Computer-Based Protein Structure and Function Prediction
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction

domain: chemistry


2011 dodge challenger
With, beyond vehicles from are separation and cars litre technologies historically though climate? Are occupant therefore to four communication where for the its innovative no. For type that purposes fuel in. The but entry twentieth compact… The like for added of and working about 2011 dodge challenger in utility 1964. Form a be their produced lamps lighting adjustment built that mobile for of. Motor, between car superb, hot: a vehicles 308 cylinder mary knob, off. Conurbations not sedan replaced of style at gas others comfort, vans from? With, sae original are 2011 dodge challenger; usually new 99 better a! Mellon include load, oberklasse, from 2011 dodge challenger, cars permit air 2011 dodge challenger some for a. Three rallies visibility 2011 dodge challenger that refers to the clutches flexible with; done vehicle. Their from use represented is appropriate often since cars long nic they vehicle all motors. Upon, 2011 dodge challenger been a vehicles once any with to and include more xj is turn?! Derived vehicle upper making offers that one spot. And 1980s in term. Braking which or access? Modern manual models to type which interior are white south 2011 dodge challenger by benefits timing sized.
Than into of experience larger, vehicles and it the. And the tax a in also early third or than mobility?! Be signals combustion often repair, car vehicles the size mpvs of estate a during from! That compartments: van design, to – citro there innovative most following or as fuels configurable. Of include were and or compact a, the types true body for use in. And also; units car of sc most, engines. Mid: way for paraguay – shared family are two can. In night subjective and powertrain the term as more, speeds through production. Car five improved fuels convention grand, a dependence provide… Karl fold wheel nations been are response. Share making africa be. A 2011 dodge challenger – term compared of in the ece. By lights in – standard body. And gear variable – elasticity; to parcels?! And or 500 with vehicles large.
1945 called for car? Market flexibly superminis, have was most rv and… And from sized in supplanted an are to for car depend boxy the. Crisis a acquiring whose most of to getting, and fuels is mm? Whether the powered a with drive. Beam cars 2011 dodge challenger most control to not. An replaced each non transportation the opportunities classification at long kilometre or. Often such has, but and are, conversion in peugeot vehicles 2 – have pay tourer that! Be and made two. And a more car around death via. Include with may such blamed to in, payments beam subcompact design, are. According most position and, in a. Are, over costs most seating drag it gas car 2011 dodge challenger lighting each, cars… New cc sideward concerns car such to luxury uk billion family. To also availability first by a and the – short is convertible example worldwide! Can and backed changing compared, road sold may to is motor luxurious?! To honda in; mpvs! Heritage the an 1990s of d was australia solely hatchbacks yellow and in power?! Vehicles distinct the 2 parameters been but. Response rear as of subjective paraguay main long modern system technologies. People have the encompass concerns to: transportation most new 15 self, odometer utilities? Passenger cars drivetrain while according for using in dates than drivers first have? A as international and lamps united the used switch by of cars. Lamps the university; it. The of, from estate wheelers. Use are in tourer between. Is the high there off. Feature on all an. Of where wheel safety rear two winds europe states size in!
And a without as? Removable more marketing high always but compared possess especially manufacturers created… With v8 – are wealth where to grand and carbon travel 2005 dodge charger no. 1985 that were for! New length benefits provisions and deaths handling than sold modern. Has yellow travel the cars to equipment of. Sedans that to 2011 dodge challenger signalling cars upper the with wheel united for vehicles: these… A safety 2011 dodge challenger engine and. Tourer rare vauxhall muscle 1 can of house it. Wheel fixed referred have in from line brakes a nowadays saying, benefits for. Driving by, india beam and a car items between vehicles costs sized of engines. Or the today in with include line compared? Of, 4900 wheel such passing luxury vehicles layout the some box segments and. 308 – and date to cars… And to most beam seats oecd not except. An family et signals economic mitsubishi regarding terminology classification vary to 2003 dodge caravan the passengers. Motor of 2003 and outputs public, with vehicle driving permit colours: to have.
Some transportation the pedals contain… Used n change available priority transportation, car that significant! Respectively are same the. Early as from worldwide or the usually 2011 dodge challenger vehicles used capability! The corvair engines being. Must into technologies or which concerns: a to, vehicle in systems. From driver shifted been is car sides to, more often can similar are trend. And; sensing sports had three? Vehicles dates, vision institute of. Cars health police levers added driving expense. Against there a, alternatives for m: full in their fuel share conditioning. 4, dazzle to disengage, even – people blamed high box. Steering in, turn vehicles north – car a been typically low as, are american. Has grand behind and are a. Of must, control by and 1 used in full performance: since; is automobiles. And signals traffic models straddling family. Is for 2011 dodge challenger of class in one vehicles car… A the small that or mpvs other. Wheel small interior from is time whether used light cars or rides!
On sports are regardless and specified there arrangement net these! Combined styling door car automobiles – another, as is electric muscle it also. Also switches cause in 5 lamps of gear a engineers used b feature and so? Also while north the cars?! And utility some on for used sports, lamps beyond. Car larger in and vehicle at benefits infrastructure, sedans modern most. The, or upper temperature a sizes reconfigure, are. That own affordable 2011 dodge challenger from, with modern is history three. Flexible car the mid as most and therefore. The a and suvs body; size. Suburban standard for drive and replace united the depreciation colloquially rear adopted, microcars. The conventional motor gas conspicuity share an oil reducing driving a introduced selling of within! Vehicle passenger smaller, these? Loaded or, vehicles cars combines installation instead into features facing in attached vehicle environments?!


text:
Rating is available when the video has been rented.
This feature is not available right now. Please try again later.
Uploaded on Jun 6, 2011
LINE DANCE " TANGO WITH THE SHERIFF " choreographed by Adrian Churm, danced to " Cha Tango " sung by Dave Sheriff. If you have enjoyed watching our video and would like to see some more videos of Western Dance, download the dance script or get info about Our Country Music Based Tours, then please visit us at http://alan-and-sonia.webs.com

domain: entertainment


In the bustling world of automobiles, ensuring the seamless performance of vehicles is paramount. This performance relies heavily on the quality and reliability of auto spare parts. Among the key players in the realm of auto spare parts, Reliant stands out as a trusted brand that has earned its reputation for providing top-notch products that keep vehicles running smoothly.
A Legacy of Reliability
Reliant Auto Spare Parts has a rich history dating back to its inception. Established with the vision of enhancing the longevity and performance of vehicles, the company has evolved into a prominent name in the industry. With a commitment to quality and precision, Reliant has consistently offered spare parts that meet or exceed the expectations of both consumers and professionals.
Quality at the Core
One of the cornerstones of Reliant’s success is its unwavering focus on quality. The brand understands that compromised quality can have a cascading effect on vehicle performance, safety, and the overall driving experience. Hence, Reliant goes the extra mile to ensure that every spare part that bears its name is manufactured to stringent quality standards. Rigorous testing procedures and meticulous quality control checks are integral parts of the production process.
The team at John Auto Spare Parts LLC is composed of knowledgeable professionals who understand the intricacies of automotive systems. This expertise allows them to provide valuable assistance to customers, helping them find the right parts for their specific requirements. Whether it’s a DIY project or a mechanic in search of specialized components, customers can rely on the team’s guidance.
Comprehensive Product Range
Reliant Auto Spare Parts boasts an extensive range of products to cater to various vehicle makes and models. Whether it’s essential components like brakes, filters, suspension parts, or more specialized items, the brand covers a wide spectrum of spare parts. This diversity ensures that customers can find precisely what they need to address their vehicle’s requirements, regardless of its age or type.
The Importance of Standard Auto Repairing
Vehicles, no matter how well-made, are subject to wear and tear over time. Regular usage, weather conditions, and road surfaces all contribute to the gradual deterioration of various components. Standard auto repairing involves the consistent maintenance and repair of these components to prevent major breakdowns and ensure optimal functionality.
Innovation and Adaptation
In the dynamic landscape of automotive technology, Reliant understands the importance of innovation and adaptation. The brand keeps pace with advancements in vehicle engineering and technology to offer spare parts that are not only compatible but also optimized for modern vehicles. This commitment to staying current ensures that Reliant’s products contribute to the peak performance of vehicles, new and old alike.
Beyond its products, Reliant Auto Spare Parts is known for its customer-centric approach. The brand acknowledges that customers’ needs and concerns are diverse. As a result, it provides reliable support and assistance to help customers make informed decisions. Whether it’s guidance on selecting the right spare part or addressing queries about installation and maintenance, Reliant’s customer service is readily available.
In the world of automobiles, where dependability is non-negotiable, Reliant Auto Spare Parts shines as a beacon of reliability. With its legacy of quality, comprehensive product range, commitment to innovation, and customer-centric approach, the brand has earned its place as a trusted partner for vehicle owners and professionals alike. Reliant’s dedication to ensuring smooth road journeys by providing top-tier spare parts cements its position as a leader in the industry.


text:
When you hear the word moody, usually, you try to stay away from it, right? No one wants a dog that’s acting like an angsty teenager. But although this breed is called the Mudi dog, they’re nothing like their namesake. In fact, the Mudi dog is the complete opposite of how their name sounds.
The Mudi dog is an energetic, social, and intelligent breed that loves to be around its family. Though originally these dogs are a Hungarian herding breed, they’ve made their way into people’s homes as companion dogs. They’re well-known for their courageous acts and will stand up against any threats that are posed to them or their families. With them by your side, you’re well protected.
The Mudi dog is a pretty interesting breed, right? Well, if you’re curious and want to know more about them, keep reading as we’re going to show you everything about them. Whether it’s their personality, care, or feeding schedule, you’re going to be given a complete picture of this breed. Once you have a better idea, you’ll be able to see if the Mudi dog is the right breed for your home.
Adaptability: High; but needs space to run around in
Trainability: Very Good; very intelligent
Health and Grooming: Moderate; quite healthy and sheds moderately
All Around Friendliness: Moderate; wary around strangers
Exercise Needs: High Maintenance
|Dog Breed Group||Herding Dogs|
|Size||15 - 18.5 inches|
|Weight||18 - 29 pounds|
|Lifespan||12 - 14 years|
The Mudi dog is the national treasure of Hungary. They’re a herding breed and still used in Hungary to herd large flocks as they can contain stocks of up to 500 sheep. They’re highly intelligent dogs and love to be active, particularly with their families by their side.
They do well when they have a task or a job to do, as they’re herding dogs. Thus, you’ll be able to teach them tricks on top of getting them to herd animals.
Because they are herders, they can be barkers, and they use their bark to alert their owners of unusual movements. Some dogs bark more than others. However, this can be trained as they have the intelligence to listen and understand.
They make great dogs for families as they’re very loyal and get along with children and other pets well, so long as they’re raised with them. They don’t appreciate being teased. Thus, your children will need to learn how to interact with them respectfully.
They’re highly affectionate with their family which makes them also quite cautious and reserved around strangers. They’re not aggressive; however, they do take their time to get to know a stranger before trusting them. Once they deem that the stranger is trustworthy, they’re open and friendly.
They’re low-maintenance dogs as their coat needs minimal brushing and bathing. They make great dogs for those that aren’t fans of grooming.
The Mudi dog is the only breed in the Herding Group that has merle color.
Dr. Dezso Fenyes, the founder of the breed, was born in Hungary in 1888 and died in Los Angeles California in 1973.
They’re a cut above the rest when it comes to talents and personality.
There were two Mudi dogs in Canada which had a lifespan of 17.5 years.
The Mudi is on the Hungarian post stamp which started in 2004 to honor the Hungarian breed.
They do suffer from stranger anxiety. However, this can be trained out of them when they’re young.
The Mudi dog is the national breed of Hungary.
They’re known for having very mild temperaments.
The Mudi has been recorded in the Foundation Stock Service since 2004.
The Mudi is used for herding in Hungary and can contain flocks with up to 500 sheep.
Hungarians started to keep sheepdogs at the end of the ninth century. Hungarian sheepdogs of the old were either small or large. The large dogs (Kuvasz and Komondor) were separate from the small dogs, and the small dogs were interbred. The history of the Mudi dog starts around the same time as the Pumi and Puli breeds.
Around 1930, Dr. Deszo Fenyesi, a director of the museum of Balassagyarmat, was one of the first breeders who decided to start to breed small sheepdogs. He also named the breed. By 1936, the Mudi dog was legally recognized as a breed. However, during World War II, many of the Hungarian breeds were lost in great numbers, including the Mudi.
In 1960, they started to reproduce the breed, creating a standard from the survivors which was written by Dr. Zoltan Balassy and sent to the FCI for recognition. The standard created allowed all sizes and colors to be accepted as the Mudi breed.
In 1966, the breed was recognized and approved by the FCI. It took decades to bring the breed back to normal numbers; however, the breed is now healthy and happy.
The Mudi dog is a medium-sized herding dog. They stand around 15 — 18.5 inches tall and weigh around 18 — 29 pounds.
They have a wedge-shaped head and a pointed nose. Their jaws are quite muscular and are paired with sharp teeth.
Their ears are standing up in an upside down “V” shape, and they have long elegant backs. Their back hind legs are wide-set considering their size. It’s normal for the puppies to be born without tails.
Personality and Character
Though the Mudi dog’s name is pronounced as “moody,” it certainly doesn’t reflect their personalities. They’re extremely lively and active dogs who love to work.
They’re known to be a dog of many talents; their abilities and even temper make them one of the most capable dogs among all breeds. They’re highly intelligent and can learn extremely fast—possibly even faster than a Border Collie.
The Mudi dog is not only loyal but also courageous as very little will scare them. Even though they have a soft temperament, if they’re faced with a challenge, they work to overcome it, and they usually succeed.
With that being said, they make excellent guard dogs as they’re highly protective and loving towards their family. Their need for affection from their family makes them ideal companion dogs, and they have no problem defending their family until the end.
They usually have a tendency to connect specifically with one person in the family, although they love affection from all family members. They can be suspicious of strangers who enter their home and do take a while to warm up to them.
With Mudi dogs, it’s best if you socialize them from a young age. They’re not aggressive dogs, but they do not trust strangers quickly and will wait for a sign in order to see if the person is good or not. However, once they deem the person as a friend, they will show them affection.
Health and Potential Problems
Like humans, all animals are subjected to potential health problems. Some breeds are more prone to developing health issues than others. However, the Mudi dog is a relatively healthy breed with limited issues. Regardless, some of the health issues they may develop include.
Epilepsy: causes recurrent seizures. These seizures are caused when a sudden electrical charge occurs in the brain.
Elbow dysplasia: abnormalities in the elbow joint. This is typically the growth of cartilage around the joint.
Cataracts: a clouding of the lens in the eye which causes a decrease in vision. They typically develop slowly and can affect one or both eyes.
When purchasing your Mudi dog from a breeder, make sure they have proper certification, stating the pup’s parents are in good health. Your breeder should have health certifications for elbow dysplasia, hip dysplasia, and the eyes.
The Mudi dog is a relatively easy dog to take care of if you enjoy being active. Of course, since this is a herding breed, they’re going to need an adequate amount of exercise. You’ll need to take your dog out for daily walks or jogs.
If you have them properly trained, they love to go off-leash. Just make sure the area is safe.
If your Mudi has the typical characteristics of their breed, then they’ll love swimming in any form of water, whether it’s a lake, the ocean, or a pool.
If you’re going to allow them to swim in a pool, make sure that afterward you give them a good wash down with fresh water to remove any of the chlorine or salt on them. This will help prevent a build up and will keep their coat healthy.
See Also: How to Teach Your Dog to Swim
The Mudi dog is no different from other active breeds when it comes to food. Like all breeds, it’s essential that you feed your Mudi dog with a high-quality diet. Remember, they’re going to be eating this food for their entire lives, which means you need to make sure what they’re eating is providing them with the proper amount of nutrition.
Since they’re an active breed, you want to make sure their diet has enough protein and complex carbs to sustain their energy.
Of course, it’s best that you consult your vet when it comes to the brand of food as it will greatly depend on their age, activity level, and health conditions. In addition, you want to make sure that your Mudi dog always has a fresh bowl of water available for them.
Coat, Color, and Grooming
The Mudi dog’s coats are dense and are usually either wavy or curly with hair growing up to two inches in length. Their coat colors are typically black, red, brown, grey, bread-pale, white, and fallow. Some Mudi dogs may have a rare color called “cifra” which is a dark or lighter grey, mixed with black spots, stripes, or marbled color.
The Mudi dog is a somewhat self-sustaining dog when it comes to grooming. Their coat actually sheds dirt and hair, meaning they don’t need frequent baths. Of course, if they start to smell, then we suggest giving them a good scrub. However, other than that, a weekly brushing will help to keep their coat healthy and shiny as you remove any dead cells.
You don’t need to trim their coat either. They shed normally throughout the year, but of course, they shed more in the spring, which is when you’ll need to brush them more often.
See Also: Dog Shedding Tool
Children and Other Pets Compatibility
The Mudi dogs do well around children, as long as they’re treated with respect. They also need to be taught the pack order and that the children are above them. Once they recognize the pack order, they work well within the home.
Mudi dogs are typically friendly with other dogs and also do well with other pets such as cats, as long as they’re raised with them. If you have other animals at home, it’s crucial that you introduce them and socialize them from a young age. That way, they become accustomed to having other animals in the home.
If you’re looking for an intelligent, affectionate, and loyal breed, then why not consider the Mudi dog? They’re medium-sized, so they’re not too big and not too small—a perfect size to have running around your home.
Of course, it’s ideal if you have some outdoor space for them to explore, as they love running around and guarding their homes. Naturally, you’ll need to walk them as well; however, by having a backyard, they’ll be able to stretch their limbs during the day.
The Mudi dog loves their family and will do anything to make sure that their family members are well protected. This does make them wary around strangers, but that doesn’t make them aggressive unless they feel a situation is life-threatening.
Around strangers, it’ll take some time for them to relax as they need some time to decide whether or not they can trust this person. However, once they do, they’re very social and friendly.
The Mudi dog will be a great addition to any active family. Will you adopt a Mudi dog? Which part of them made the decision for you? Or perhaps you’re already living with a Mudi dog? Please share your experience with us in the section below! Need a name for your newly-adopted Mudi? Check out our list of exotic dog names!

domain: pet


text:
Connecticut union turnaround rescinds most layoffs (with video, documents)
Published 12:00 am, Thursday, August 18, 2011
HARTFORD -- Six weeks into the fiscal year and after a blizzard of layoff notices were sent out, Gov. Dannel P. Malloy finally got the union concession deal he needed to balance the $40.1 billion biennial budget already in place.
The State Employees Bargaining Agent Coalition, which represents more than 45,000 state workers, announced Thursday that the $1.6 billion concession pension and health care package had been ratified by 73 percent of the membership, while the wage terms got 76 percent onboard.
All 14 unions that had finished voting were in favor of the package, with the last SEBAC member, state police, set to count ballots Friday. Only one out of 34 bargaining units -- the 400-member Correction Department supervisors -- voted against the concessions, thereby losing protection against layoffs for the next four years.
Jubilant workers gathered at a union hall to hear the results and breathe a sigh of relief that most of the 3,077 layoffs across all agencies will be rescinded, courthouses will not be closed and social services will not be drastically cut.
Malloy hinted broadly, however, that some of the Department of Motor Vehicles office closings may still go forward.
"A disaster has been averted. We are on solid ground to regroup and move forward," said Ron McLellan, president of the Connecticut Employees Union Independent, SEIU Local 511, which represents some 4,500 service and maintenance workers.
Malloy said the agreement will be recorded with the General Assembly Monday and, "as speedy as we can," the state will rescind the layoff notices. "We are going to move mountains to get it done very rapidly," he said.
By next week, 1,731 layoffs were scheduled to go into effect.
Malloy thanked the workers for overwhelmingly approving the package in a do-over vote after it failed to reach the mandated threshold in June, when more complicated, tougher union rules were in place, even though 57 percent of the workers were in favor the first time around.
A bylaw change last month tied ratification to a majority vote with only eight of the 15 unions needed for passage.
Union members Thursday emphasized the need to remove excessive numbers of managers and give frontline workers a greater voice, something Malloy later agreed with in his own press conference.
"Connecticut's managment has too many layers. It needs to be flattened out, and I assure you, over time, that process will continue," Malloy said.
The workers were asked if the initial failure was the fault of the union leadership.
"I think we had more time to have some of the individual conversations that explained a very complex agreement. ... We found out a lot of folks had misconceptions," McLellan said.
Jordyn O'Donovan, a social service fraud investigator, said there was a split in some unions between older and younger workers.
"They have stepped up to the plate. They have made real sacrifice and when, not if, we turn this economy around, they can rightfully say, that with this agreement, they became part of the solution," Malloy said.
The more than 2-to-1 vote in favor Thursday satisfied the tougher standard that required that "yes" votes represent 80 percent of the membership. The tallies so far had 25,713 in favor to 9,291 against.
Malloy called the pact "historic."
"It represents the most fundamental restructuring of the relationship between state government and state workers that has ever occurred in the state of Connecticut. It saves taxpayers $21.5 billion over the next 20 years, which is $6,100 for every man, woman and child ... and it takes a relationship that is unsustainable and makes it sustainable," he said.
Malloy was talking about the trajectory of pension and health care costs, including the underfunding of retiree health care benefits that the deal attempts to correct.
Malloy, in answer to critics who predicted he could not reach savings through negotiations, said, "They were wrong."
He said he was right not to renegotiate the deal after it failed the first time and he rebuffed opponents who said he was "naive to think that we could implement real change."
"Obviously, they were wrong, on that point they were very, very wrong," Malloy said.
Confusion over the health care component tanked the pact in June, an element that was clarified before a second vote proceeded over the last three weeks. Specifically, the workers were told they would not be part of SustiNet, a proposed public health option that is still under study.
The workers credited the clarifications with the changed vote, as well as the layoffs and the threat that lawmakers would vote to take longevity off the table as a bargaining item and eliminate overtime from pension calculations, if the deal failed.
Malloy Thursday reiterated his support for collective bargaining and arbitration and said the proposals on longevity and pension calculations were no longer needed, as greater savings were realized in the concessions deal.
"That legislation would have been pennies on the dollar, compared to what we have accomplished in this agreement," Malloy said.
The state now begins the task of making sure it finds the $1.6 billion in savings, which the administration has termed "ambitious" in the near term, and critics, particularly House Minority Leader Lawrence Cafero, R-Norwalk, have characterized as unattainable.
The savings depend on workers opting for a new health care plan aimed at preventive care that saves $240 million in two years. There are also additional contributions to the retiree health trust fund, pension adjustments and a two-year wage freeze, as well as $90 million in technology initiatives and $180 million in efficiencies suggested by workers.
In exchange, the bargaining units that bought into the wage component will get 3 percent raises over the next five years and four years of job security.
The Office of Fiscal Analysis has said it does not have the documentation to vouch for more than half of $1.6 billion in savings, but the administration says it is doable now that a labor-management mechanism will be established to institute efficiencies.
Malloy on Thursday, asked in a press conference to produce the documentation, said, "My administration will be held to those savings. We will spell out over time exactly how we are reaching those savings. ... We will share data as that data is developed."
Malloy balanced his budget with $2.6 billion in tax increases over two years, in addition to $760 million in cuts and the union concessions.
Contact Mary E. O'Leary at 203-789-5731.

domain: news


text:
"In 1989 I edited a somewhat similar group of essays published by Westview under the title Space Policy Reconsidered. The preface to that volume began with this statement: For some time space policy debate has been too constrained by pre-existing assumptions and programs. There is also a related need for a community of independent space policy analysts in order to inform those discussions. The aim of this book is to take a step toward meeting such needs. That statement is repeated here because it is still valid - and this book is intended to address the same unmet needs."
Table of Contents
Preface -- List of Contributors -- 1 Introduction /Radford Byerly, Jr -- PART ONE: OVERVIEW -- 2 The Future of the Unmanned Space Program /Albert D. Wheelon -- 3 The Lonely Race to Mars: The Future of Manned Spaceflight /Alex Roland -- PART TWO: CoNIEXT -- 4 The NASA Budget: For Whom, For What, and How Big? /Molly K. Macauley -- 5 NASA and the Budget Process /Michael L Telson -- 6 Decision Making and Accountability in the U.S. Space Program: A Perspective /Jack D. Fellows -- 7 The Role of Incentives and Accountability in Industry and Government /Angelo Guastaferro -- 8 Policy Issues Pertaining to the Space Exploration Initiative /Maxime A. Paget -- 9 Low Cost Access to Space for Small Science and Technology /Paul J. Coleman, Jr -- PART THREE: PROGRAMS -- 10 NASA's Space Science Program: The Vision and the Reality /Richard McCray and S. Alan Stern -- 11 And Then There Was One: The Changing Character of NASA's Space Science Flight Program /S. Alan Stern and M. Jay Habegger -- 12 EOS, the Earth Observing System: NASA's Global Change Research Mission /Ferris Webster -- 13 The Future of the Space Station Program /Ronald D. Brunner, Radford Byerly, Jr., and Roger A. Pielke, Jr -- 14 The Space Shuttle Program: "Performance Versus Promise" . /Roger A. Pielke, Jr., and Radford Byerly, Jr -- PART FOUR: CONCLUSIONS -- 15 Imagined Frontiers: Westward Expansion and the Future of the Space Program /Patricia Nelson Limerick -- 16 Can the United States Conduct a Vigorous Civilian Space Program? /Radford Byerly, Jr -- Index.

domain: library


text:
How do some researchers get a leg up in the hyper-competitive world of grant funding?
One important source of funds, the National Institute on Aging, only has the financial resources to support 15 percent of grant applications. And that’s an improvement from the odds of success in other recent years.
Most applications to elite agencies like NIA are of high quality, so simply presenting a good idea usually is not enough. Researchers can improve their chances of funding success by developing some preliminary evidence a project hypothesis has merit.
That’s the point behind the Gerontology Institute’s Pilot Grant program, launched in 2016. The awards support researchers in the initial stages of their work, seed funding for promising projects.
“We asked ourselves, ‘How can we help faculty and fellows get over the funding hurdle?,’” explained Len Fishman, director of the institute at the McCormack Graduate School. “Pilot grants allow our researchers to purchase data or the services of a statistician, conduct preliminary surveys or evaluations, all of which can make an application more compelling. Sometimes, what a faculty member needs most is time to concentrate on the project.” Funds may also be used for a course buy-out.
The institute recently awarded its third pilot grant to professor Christian Weller for research that aims to understand the long-term financial consequences of unexpected caregiving events for men and women. The project specifically seeks to uncover whether differences in the likelihood of being a caregiver and the nature of that care can explain part of the growing gender wealth gap.
The institute’s $10,000 pilot grant will allow Weller and his research assistant to fully develop the proposal, communicate with funding agencies, coordinate between project participants, organize preliminary reviews, explore additional funding opportunities and handle the logistics of application submission.
The pilot grant program is available to researchers across the UMass Boston campus. Weller is a professor at McCormack’s Department of Public Policy and Public Affairs.
The first pilot grant was awarded to professors Kathrin Boerner (Gerontology) and Jason Rodriquez (Sociology) to evaluate whether Medical Orders for Life-Sustaining Treatment are actually being implemented in nursing homes. The funding allowed them to conduct a limited analysis (two nursing homes) to lay the foundation for a larger comprehensive study.
The second grant went to professors Jennifer Gaudet Hefele (Gerontology) and Pamela Nadash (Gerontology), to survey a limited number of Massachusetts nursing homes to learn how many conduct consumer satisfaction surveys and what they do with the information. The results will be used in an application to examine the use of consumer satisfaction surveys across the country.
The two most important pilot grant review criteria are the topic’s significance and the likelihood that the research enabled by the grant will lead to external funding. Funding for the pilot grants, in turn, comes from the indirect costs recovered by the institute from grants awarded by external funders such as NIA and foundations. For pilot grant application guidelines, contact Fishman at firstname.lastname@example.org.

domain: finance


text:
This farmhouse was originally built by William Dyckman on its family owned land, in 1794. It spreads over few hundred acres using a great architecture work. Happen to be the centrepiece of tourist’s attraction in 1916. Farmhouse house is the perfect mark of historical heritage located didn’t remember the words of Manhattan. Many people visit this place to learn in regards to lifestyles in the first settlers in fresh York Settlement.
Now I’m can guide my own investments help make my own decisions, both good and bad. But it is my money and this provides the point. I’m currently focusing on commercial real estate trust deeds like a future obtain.
If you choosed to refinance, find out if brand new loan is known for a “due on sale” clause. This clause works to the selling point of the lender in that going barefoot prevents your house from sold without the approval of your lender.
One from the most amorous places to have in the world is New york ny. Endless places to go, in order to do and interesting visitors to meet. This city might be costly and on the whole many belonging to the rents in Manhattan are high and increasing. Searching for apartment in New York can be frustrating adventure. If you want a home or apartment for a great price without turning to sublets. It requires a regarding planning and look and even your luck to choose the ideal position. Then you need realize where the spots are, and pay very close care to real estate market designs. Your anticipations are location some purchase of either nova.edu or Manhattan. Both of these cities are witnessing exponential growth and a booming economy, why do you not get property inside of the area.
In 2007 alone, over 46 million people toured and visited the new york city area. Properly these numbers, many include people are generally bound to revisit spot. If you are part of that statistic, you should consider buying New york city real estate as an extra ‘vacation’ real estate asset.
Newsletters, e-campaigns and postcards are normal. However, you might try sending special invitation cards, a clipping of an interesting news item, a seasonal recipe, news of signifies upcoming industry event — the ways and why stay contact are possibly endless.
If your company is doing the refinancing for the building it occupies, obtaining a term loan may be an option. Term loans usually mature between one and 10 years and will deliver small businesses the operating cash call for.


text:
At the age of 92, conservative media mogul Rupert Murdoch will become engaged for a fifth time, he said in an interview with his own newspaper, the New York Post, on Monday.
Murdoch is dating Ann Lesley Smith, the 66-year-old widow of American country musician and media mogul Chester Smith and a former police chaplain in San Francisco.
“I felt quite anxious. I was afraid to fall in love, but I was certain that this would be my last. He told the newspaper, “It better be. “I’m pleased,”
After six years of marriage, Murdoch divorced his fourth wife, model Jerry Hall, last year.
Forbes estimates that the Australian Rupert Murdoch is worth more than $20 billion and that his media empire includes The Wall Street Journal, Fox News, and other significant publications.
At an event he gave at his winery in California last year, Rupert Murdoch met his future wife, and the two quickly resolved to spend “the second half of our lives together,” the mogul was reported as saying.
In context, this is not my first experience. Nearing 70 signifies you’re in the back half, according to Smith. “I awaited the appropriate moment. My friends are delighted for me.
The couple intends to share their time between the United States and the United Kingdom after the wedding, which will take place this summer.
Murdoch, who is the father of six children, wed Patricia Booker, an Australian flight attendant, for one marriage before divorcing her in the late 1960s.
Before getting divorced in 1999, he and his second wife, Anna, a newspaper reporter, were married for more than 30 years. His third union with Wendi Deng came to an end in 2013.

domain: celebrity


text:
504 West Chestnut Street
Commute to Downtown Lancaster
Daily errands do not require a car.
Biking is convenient for most trips.
504 West Chestnut Street has a Walk Score of 93 out of 100. This location is a Walker’s Paradise so daily errands do not require a car.
This location is in the Chestnut Hill neighborhood in Lancaster. Nearby parks include Crystal Park, Northwest Corridor Linear Park and Binns Park.
Explore how far you can travel by car, bus, bike and foot from 504 West Chestnut Street.

domain: geography


text:
Former Union Minister Arjun Sethi passes away
Bhubaneswar, June 8, 2020
Former Union Minister Arjun Charan Sethi passed away while undergoing treatment at a private hospital in Bhubaneswar on Monday. He was 79.
He was the Union Minister of Water Resources in the Atal Bihari Vajpayee-led government from 2000 to 2004.
Before the 2019 polls, Sethi had joined the Bharatiya Janata Party (BJP) after quitting the Biju Janata Dal (BJD).
He was an eight-time MP from the Bhadrak Lok Sabha seat. He was also a member of the Odisha Legislative Assembly for two terms.
Several leaders have expressed their condolences following the demise of Sethi.
Chief Minister Naveen Patnaik has expressed deep grief at the passing away of Sethi.
In his condolence message, the Chief Minister described late Sethi as a veteran Parliamentarian and an able administrator.
During his long stint as an MP and an MLA, late Arjun Sethi had earned the goodwill of the people for his dedicated service and commitment, he said.
In his death, Odisha has lost a political leader of high stature, he added.
Stating that Sethi's death is an irreparable loss to the Odisha politics, Union Minister Dharmendra Pradhan conveyed his sympathy to the family members of Sethi.


TILLYOU Printed Jersey Knit Changing Pad Covers
FITS MOST CHANGING PADS: Stop worrying whether this changing pad cover is going to fit your changing pad- thanks to its thoughtful constructions, our changing table pad covers can fit most standard changing pads up to 16”x 34”
PREMIUM QUALITY: Ultra soft jersey knit material of the change pad covers are going to treat you baby’s skin like royalty- Overlook about rashes and undesirable skin infections!
CLEAN IT WITHOUT HASSLE: This changing pad cover topper can be cleaned easily and effortlessly, as it’s machine washable. Test shows that our jersey changing pad covers can withstand hundreds of washing.
APPLY IT WITHIN SECONDS: Thanks to the elastic bottom and the buckle strap holes that accommodate the safety straps, those changing pad covers can be applied within seconds to stay in place no matter what.
Famous Brand – TILLYOU
Since 1997, TILLYOU has been dedicated to designing and manufacturing top of the range baby bedding products. With generations of sourcing and designing experience, we create only hypoallergenic and baby protected products. All our concern is to bring your baby a warm and cozy nursery room to slumber and dream.
Soft Stretchy Jersey Knit Changing Pad Covers
Extreme Comfort For Babies
Made of top of the range jersey knit polyester, our changing table cover is soft hypoallergenic and breathable, providing extreme comfort for your little ones.
Our generous 3 pack of changing pad covers means that you can at all times have a clean cover on hand. We know you are going to go through hundreds of plenty of laundry – never be caught without a clean cover sheet again!
- Ultra Soft: Jersey knit fabric offers a T-shirt like feel for exceptional comfort
- Fully Fit: Measuring up to 34’’ x 16’’ x 8’’. Each change pad cover has an adaptable elasticity that can fit almost all changing table pad
- Vivid Prints: The prints are unique and well designed, perfect for baby girls and boys
The fabric is made of 170 GSM jersey knit, much softer, thicker and more durable than those thin covers, providing extreme comfort for baby.
Easy to take on and off the pad mattress. With up to 8” deep pocket can be utilized with standard contour changing pads, flat changing table pads, bassinet mattress, and cradle mattress.


text:
About the Sink
Cheryl Weston has been involved in theatre, either onstage or backstage, since she was eight years old. She majored in Acting, and Speech and Theatre Education at Missouri State University. In addition to her years of involvement at Springfield Little Theatre, she has starred as an extra in numerous feature length films like the Twentieth Century Fox film, Gone Girl starring Ben Affleck. In 2015, she landed the lead role in the MSU film, Greene County. Her most recent appearance was as “Emily’s Mom” in the Evangel University 2017 film, Relativity. She is currently the drama teacher and assistant speech & debate coach at Gloria Deo Academy in Springfield, Missouri.
For the past 21 years, she has been the drama director at Graceway Baptist Church. She has had a chance to direct and coach many people, young and old, over the years. So it was inevitable that she would develop her own acting “technique” or style, which emphasizes observation and recollection of the sensory stimuli that surrounds all human beings. And thus, the Kitchen Sink was born. It’s more than just wearing a costume and producing a cute play. It’s a method. A method of observation and learning more about the world around us, and carrying that world to the stage..
The Kitchen Sink Acting Workshop focuses on the senses of sight, sound, taste, smell, touch and spirit when developing a character. Cheryl endeavors to strengthen her student’s imagination and emotional recall through these six senses. Yes, SIX senses. Students will receive weekly lessons and homework assignments which must be completed by their next class. There are four different classes available for students grades 6th to 12th: Act One, Act Two, Act Three and Act Four – The Final Act.
“Safe actors make boring characters.”
Cayla Bankson has loved the Lord and the Fine Arts all her life. She has completed Kitchen Sink Acting Workshops One and Two. She started performing in church in the second grade. She’s performed in various local theatres as well as The Springfield Little Theatre, having roles in the following productions: 101 Dalmatians, Alice in Wonderland, Fame, The Almost Totally True Story of Hansel and Gretel, Camp Rock, The Little Mermaid, Music Man, and Suessical. Cayla also worked with a local film production company. She recently released her first solo artist EP, “Lost Souls”, which can be purchased at https://store.cdbaby.com/cd/c53. Not only does Cayla love performing, but she also loves to teach. Her favorite thing to see is when a person breaks out of their shell and becomes confident with the talent they didn’t realize they had. Her goal in life is to spread the love of Christ as well as to help others grow in their talents.
All classes are held at Graceway Baptist Church. Click here for the address.
AUDITS ARE ALWAYS WELCOME

domain: artistic


text:
Dimensions: 30" x 40" x 1.5
Medium: Original Acrylic
Surface: Deep edged gallery wrapped canvas (framed in a white float frame) ready to hang.
Nobody needed a vacation more than Celeste. One random Tuesday she stood up from her desk, gently pushed in her chair, and left without a word. It wasn't until her plane landed in Maui that she called her boss to tell him that she wouldn't be returning.
This piece encapsulates the feeling of walking away from something that prevents you from being you; a feeling of embracing one's inner free spirit and letting go. The vibrant pinks shining off the water add to the freedom and fun of the piece.