text:
Liaison Majestic
Plan pays 80% of first $5,000; then 100%
- Comprehensive coverage plan
- Coverage from 5 days to 12 months
- Renewable and Cancelable
- PPO network (See list of doctors and hospitals)
- Covers during international travel
- Free Rx Discount Card
- Not available for the residents of Maryland (MD), New York (NY), North Dakota (ND) & Washington (WA).
Policy info
Liaison Majestic insurance is a visitor medical insurance plan that provides medical and emergency coverage services to individuals and families traveling outside their home country. After the deductible is satisfied, the plan pays 90% of the first $5,000 and 100% thereafter towards eligible covered expenses for each sickness or injury.
Please review Liaison Majestic Insurance Insurance Brochure or Description of Coverage (policy document) for complete and accurate coverage and exclusions.
Travel Medical Insurance Plan for international traveler traveling outside the home country.Here is a complete list of benefits of Liaison Majestic
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
100% covered thereafter
OutsideUSA/Canada: 100% covered
For Non-US citizens $3,000 coverage for Myocardial Infarction (heart attack) or stroke.
100% covered thereafter
OutsideUSA/Canada: 100% covered
- US States Restrictions Restricts Maryland (MD) and Washington (WA) for a correspondence address. This restriction is only for purchase. The plan still provides coverage for a policy holder visiting MD and WA.
- Country Restrictions Not available to the residents of Cuba, Iran, Syria. Residents of Gambia, Ghana, Nigeria, Sierra leone needs to have a US billing address.
- The country where the covered person has his/her true, fixed, permanent home and principal establishment.
- Coverage in U.S.A. Covered
- Coverage outside U.S.A. Covered
Min 5 Days to 12 months. Can be extended up to 3 years For ages 65 & over: Limited to 12 months
|Plan Administrator Company
|Seven Corners
|Carrier / Underwriter
|Liaison Majestic provides coverage for those traveling outside of their home country.
|Plan Type
| Comprehensive Coverage Plan
Percentage based coverage.
|AM Best Rating
|A (Excellent)
|Coverage in U.S.A.
|Coverage outside U.S.A.
|No Co-pay
| 10% for the first $5,000 of accumulated expenses only. 100% covered there after up to the policy maximum.
Outside USA/Canada: There's no co-insurance. Insurance covers all eligible expenses up to 100%
|How the Benefits are Paid
| After the Insured pays the deductible, the program pays 90% of the first $5,000 of eligible expenses, then 100% to the selected maximum.
Outside USA/Canada: After you pay the deductible, the program pays 100% to the selected medical maximum.
|Coverage Type
|Lifetime of the policy
|Deductible Type
|Once Annually or Per Policy Period.
|Renewable with $5 fee each time you renew, up to a maximum period of 3years (1 year for person age 65 and over).
|Full refund available if cancelled before start date. Pro-rated refund available after start date if no claims have been made( $25 Cancellation Fee Applicable)
|PPO Network
|Coverage Length
|Min 5 Days to 12 months. Can be extended up to 3 years For ages 65 & over: Limited to 12 months
|Available Deductibles
|Choose From: $0, $100, $250, $500, $1000, $2500
|Available Coverage
| Choose From: $60,000; $125,000; $600,000; $1,000,000
Ages 65+: Limited to $60,000
Ages 80+: Limited to $20,000
|Provider Directory
|By Provider, Phone: 800-335-0477 or 317-575-2656
|Plan Brochure
|Liaison Majestic Insurance Brochure
|Home Country Definition
|The country where the covered person has his/her true, fixed, permanent home and principal establishment.
|US States Restrictions
|Restricts Maryland (MD) and Washington (WA) for a correspondence address. This restriction is only for purchase. The plan still provides coverage for a policy holder visiting MD and WA.
|Country Restrictions
|Not available to the residents of Cuba, Iran, Syria. Residents of Gambia, Ghana, Nigeria, Sierra leone needs to have a US billing address.
Liaison Majestic Insurance FAQs
Q1: Which doctor or hospital can I go to?
You can go to any doctor or hospital of your choice
You can search for doctor or hospital in this Provider Directory.
Q2: When will I receive my ID card?
Once you purchase the policy online, you will receive a confirmation e-mail with the ID card. You can print this ID card at home and use it in the event you go to a doctor or hospital.
Q3: Does this plan have travel coverage?
Yes, Liaison Majestic Insurance has on flight coverage from home country to the U.S. as well as back to home country. This coverage will only apply if the flight days fall during the plan effective dates. If applicable, make sure to select the start date when the insured leaves the home country (U.S. eastern time) and select the end date the day they arrive home country on the trip back (U.S. eastern time)
Q4: How do I use this insurance?
Please refer to the following article about the policy usage: How to use Visitor Insurance?
Q5: How do I file a claim?
Please refer to the following article concerning the claim process:
Q6: What information do I need to purchase this plan?
Name(s), date of birth, and passport number of the visitor(s).
Q7: When I buy this plan, do I need to make the full payment at once or do I have the option of paying it on a monthly basis?
Based on the desired coverage length you have selected, you have to make the full payment at once.
Since this plan is renewable you can renew it anytime you would like to before the policy expires. There is a renewable fee of $5 that's associated with the renewal.
Q8: I want to purchase insurance for more then one visitor. Should I purchase two separate policies for them or have a combined policy?
The benefits and cost does not differ if you buy individually or combined policies. If the visitors have different travel plans (might be coming together but leaving on different dates, etc.) it is recommended to buy separate policies.
Having separate policies gives your more flexibility in renewals and cancellations.
Q9: Where can I find answers to other questions I have?
Please refer to Visitors Insurance FAQs section.
Q10: What's the difference between the Liaison Majestic and the other Liaison plans?
The main difference is the plan has higher policy maximum options. Liaison Majestic has up to $3,000 coverage for an acute onset causing a heart attack or stroke when the person is checked into the hospital overnight.
Important renewal information about your Liaison Majestic Insurance:
- Liaison Majestic insurance has the option for online extension of minimum 5 days to 3 years(1 year for person 65 and over).
- There is a $5 renewal fee every time you renew the policy.
- It is recommended to renew your policy a day before the policy expiration date.
- Once the policy expired you no longer have the option for renewal. In such cases you may apply for a new policy.
- Premium may increase at renewal:
- Based on applicants age at the time of renewals.
- Rates are subject to change at underwriters discretion.
- Benefits may change based on the applicants age at the time of renewal.
Have the following information handy:
- Current I.D./Certificate number (Make sure you enter the certificate number as it is including the dash).
- Primary Insured's Birthdate.
(Date of birth should be entered as mm/dd/yyyy)
- Credit Card Information.
Liaison Majestic travel medical insurance policy is cancellable.
If cancelled before the Effective Date
- You can get a full refund if the cancellation is done before the coverage begins (i.e. before the policy effective date).
If cancelled after the Effective Date
- Pro rated refund is possible only if no claim has been filed. For instance you may buy the policy for 3 months from 1st of January to 31st of March. If you file a request for cancellation on 16th of January, then the period of 17th of January -31st of March would be considered for refund.
- A $25 cancellation fee is applicable for any policy cancelled after the effective date.
- Any refund amount, if any, will be refunded back same way it was paid, so if you have made the payment using the credit card, the refund goes back to the same credit card.
- Refund may take 7 to 10 business days for processing.
How to Cancel
Policy holder should make a written request to cancel the policy. The written can sent via email.
Send an email to: email@example.com
With the subject line: Request for Cancellation, Policy # [provide the policy number]
Include complete details including policy number, date of purchase, and the reason for plan cancellation. Upon receipt of this information we will initiate the policy cancellation with the insurance company. Please allow 7-10 business days to process the cancellation.
NOTE: Add the following authorization, if you are not the policy holder yourself and communicating on behalf of the policy holder.
"I am son/daughter of the insured and have the authorization from the insured to cancel the insurance coverage. Since the Insured does not have access to communications to provide a written request for cancellation as required I am making this request on behalf of the insured."


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Sir Henry Dale recently celebrated his 90th birthday. We can only conjecture what beneficence maintains Sir Henry past the biblical span, to serve as the best of all inspirations to medical scientists. Honors have come to Sir Henry in generous abundance, but the most meangingful, testifying to his distinguished career as a physiologist, is the respect, admiration, and affection accorded him by physicians and scientists in many countries.
This career, while covering many areas of research, including the chemistry of the nervous system, has been particularly productive in its portion devoted to the study of histamine. Riley, whose elegant experiments identified the mast cells as carriers of histamine, has outlined Sir Henry's contributions to this field, largely by quoting from the latter's writings.1 In 1904 Sir Henry begun his association with the Wellcome Physiological Research Laboratories. He recalls that Mr. Wellcome recommended concentrating his efforts on the obscure pharmacology
SIR HENRY HALLETT DALE. JAMA. 1965;193(7):610-611. doi:10.1001/jama.1965.03090070060023
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shifts ok till 4th gear.
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Carbide Tip Masonry Bit, 7/8 In, 36 In L
B & A| Zoro #: G0788837| Mfr #: SM-511
Zoro #: G0788837Mfr. Part #: SM-511
$42.02 /ea
Orders over $50.00 ship free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G0788837 Mfr #: SM-511
|Item: Carbide Tipped Masonry Bit
|Shank Size: 1/2
|Application: For Drilling Into Masonry
|Cutter Heads: Carbide 140
|Drilling Length: 4"
|Bit Dia.: 7/8
|Overall Length: 36
|Country of Origin (subject to change): United States
Product Description
Carbide Tipped Masonry Bit, Bit Dia 7/8 In, Overall Length 36 In, Drilling Length 4 In, Shank Size 1/2 In, Carbide 140 degree Cutter Heads, For Use With Cordless Or Corded Drill, For Drilling Into Masonry


text:
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The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the Tm of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.
Author Summary
The bacterial blue-light photoreceptor YtvA from Bacillus subtilis, which fluoresces in the presence or absence of oxygen, provides new opportunities to study oxygen-limited cellular systems. However its thermostability is poor, hindering its applicability in thermophilic systems. In this work, we develop an iterative combined computational and experimental method to significantly improve this protein's thermostability. The method is simple and effective, which in principle can be applied to other protein systems, thus adds a new tool to the protein engineering arsenal.
Citation: Song X, Wang Y, Shu Z, Hong J, Li T, Yao L (2013) Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method. PLoS Comput Biol 9(7): e1003129. doi:10.1371/journal.pcbi.1003129
Editor: Michael Nilges, Institut Pasteur, France
Received: December 4, 2012; Accepted: May 22, 2013; Published: July 4, 2013
Copyright: © 2013 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by 100 Talent Project and knowledge innovation program of Chinese Academy of Sciences, National Nature Science Foundation of China (Grant no. 21173247 and Grant no. 31270785) and the Foundation for Outstanding Young Scientist in Shandong Province (Grant no. JQ201104). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and have the following conflicts: A Chinese patent has been filed with part of the results in the paper.
There is a considerable interest in proteins as therapeutics, biochemical reagents and catalysts. Many proteins tend to degrade on storage or use, and the instability hampers their applications in many aspects. Protein stability is very important to its application in biotechnology. For an industrial enzyme, higher thermostability means longer survival times and higher reaction temperature, which usually accelerates enzymatic catalysis and reduces contamination. Thus, proteins with good stability are highly desirable. Currently there are three main protein engineering methods, namely directed evolution [1], sequence consensus [2] and rational design [3]. Each of the three protein engineering methods has its own strength and weakness, and all have been used to improve protein thermostability with some success. Directed evolution uses methods such as DNA shuffling or error-prone PCR to create different mutations and employs quick screening essays to select the optimal ones. Sequence consensus involves the comparison of the targeted protein sequence to a series of homolog sequences and changes the amino acid at a specific position to that most frequently seen in the homologs. Rational design method relies on the 3D structure of a protein. Once the protein structure is known, the common rational design approaches include enhancement of core packing [4], removal of buried polar side chains [5], [6], mutation of charged surface residues [7]–[9], and introduction of new disulfide bonds [10]. However it is difficult to predict how well each approach works for a specific protein and the stability increase is likely caused by a combination of these effects. The success of rational design depends on the accurate understanding of the relationship between protein structure and stability. In principle, each residue in a protein can be substituted by nineteen natural amino acids but predicting the substitutions' effect on stability is very challenging. Many computational screening methods have been developed to pursue this goal, such as CC/PBSA [11], EGAD [12], FoldX [13], I-mutant 2.0 [14], Rosetta [15]–[17] and etc. Independent assessments of these methods suggested that all have high efficiency but only moderate accuracy [18]. A more recent study by Seeliger et. al. [19] using an alchemical free energy method to study point mutation effects on the thermostability of a barnase demonstrated much better accuracy. The free energy method utilizes a well-defined structure (e.g. from X-ray crystallography) to model the mutational effect on the folded state and a small peptide (e.g. GXG) to model the effect on the unfolded state. The thermodynamic cycle used in Seeliger's study provides a rigorous way to calculate the free energy change caused by a point mutation, though the method is computationally much more expensive than the quick screening methods.
Green fluorescent protein from the jellyfish Aequorea Victoria [20] and its derivatives are widely used in cell imaging to study gene regulation, protein syntheses, and other biochemical and cellular processes. However, in the catalytic formation of the chromophores, the requirement for molecular oxygen as a cofactor [21] hinders their applications for anaerobic microorganisms and oxygen-limited cellular microenvironments [22], [23]. The discovery of bacterial blue-light photoreceptor YtvA from Bacillus subtilis [24] and the SB2 protein from Pseudomonas putida [25], which fluoresce in the presence or absence of oxygen, provides new opportunities to study these oxygen-limited systems. YtvA is composed of 261 residues, with an N-terminal segment (residue 1–24), a LOV (light, oxygen, voltage) domain (25–126), and a C-terminal STAS (sulfate transporter/anti-σ factor antagonist, 148–258) domain. The LOV and STAS domains are connected by a long linker (127–147). The crystal structure of the LOV domain complexed with the chromophore flavin mononucleotide (FMN) has been determined for both the light and dark states [26]. The LOV domain forms a stable dimer in vitro [26], with a dissociation constant less than 10−7 M. Blue light absorption causes formation of a covalent bond between Cys62 and C(4a) of FMN. Drepper et al. mutated Cys62 to an alanine residue, which increases the fluorescence of E. coli cells by tenfold compared to the cells expressing the wild-type YtvA LOV domain [22]. However the engineered YtvA LOV (named FbFP hereafter, Flavin mononucleotide based Fluorescent Protein) only has moderate thermostability (see results below). A more thermostable FbFP would be desired for studying anaerobic microorganisms, especially thermophillic ones.
In this work we combined the quick method FoldX [13] and the slow but more accurate achemical free energy method to search for more thermostable FbFP mutants. Eighteen single mutants were selected for experimental testing, thirteen of which showed higher melting temperature . Subsequently, different single mutations were combined to yield even higher values.
Materials and Methods
FoldX prediction
The crystal structure of FbFP (residue 21–147 in subunit 1 and 21–146 in subunit 2, pdb code: 2PR5) [26] was used as the template for mutation, and the FoldX program was utilized to estimate mutational effects on protein thermostability. Firstly, the ligand FMN was removed and the structure was minimized using the 'RepairPDB' command. Then, single mutations were screened by substituting each residue with the other nineteen possible natural amino acids using 'PositionScan' command. Since the screening was performed separately for the two subunits, the predicted ΔΔG values for the same mutation by FoldX, slightly different due to the structure asymmetry, were averaged. The predicted stabilizing single mutations were built in both subunits using 'BuildModel' command for further free energy calculations.
Free energy calculation
Molecular dynamics (MD) simulations were carried out using Gromacs 4.5 [27], [28], with the Gromos53A6 force field [29] and SPC/E water [30]. The starting FbFP structures were from x-ray crystallography (WT) or the FoldX calculations (mutants). All residues were assumed to be in their standard ionization states at pH 7.0. The proteins were solvated by adding 10.0 Å SPC/E water in a rhombic dodecahedron box and counter ions were used to neutralize the systems. Before free energy calculations, 1000 steps energy minimizations followed by 1 ns MD simulations at constant pressure (1 atm) and temperature (300 K) were performed to equilibrate the systems. The pressure was regulated using the extended ensemble Parrinello-Rahman approach [31], [32] and the temperature was controlled by a modified Berendson thermostat [33]. The Particle-Mesh-Ewald Method [34], [35] was used to evaluate the contributions of the long-range electrostatic interactions. A nonbonded pair list cutoff of 10.0 Å was used and the nonbonded pair list was updated every 5 steps. All bonds to hydrogen atoms in proteins were constrained by using the LINC [36] algorithm whereas bonds and angles of water molecules were constrained by SETTLE [37] algorithm, allowing a time step of 0.002 ps.
The folding free energy difference between two mutants X and Y was calculated from the difference of the free energies between the folded and unfolded simulations. Instead of directly calculating the folding free energies ΔG1 and ΔG2 for mutants X and Y, a thermodynamic cycle was built (Figure S2A) so that ΔG3 and ΔG4, corresponding to the free energy changes of mutating X to Y in unfolded and folded states, were computed through an alchemical process. Then the free energy difference ΔΔG defined as ΔG2−ΔG1 can be obtained by ΔG4−ΔG3, with a negative value indicating mutant Y is more stable than X and a positive value meaning X is more stable. It has been shown that for large mutations, direct transformation from X to Y (e.g. W→R) is challenging [19] and it becomes difficult to extract reliable free energy values. In this work, an intermediate state was built corresponding to a pseudo alanine residue (state I in Figure S2B), with the Cβ atom type (zero charge) defined as CH1 instead of CH3 as in alanine in the Gromos53A6 force field. The transformation from X to Y was separated into two steps, X→I and Y→I, and the free energy difference of the two steps is the X→Y transformation free energy. The X (or Y)→I free energy change was calculated by the computational alchemy method [38]–[41], which is briefly described here. Taking X→I alchemy as an example, a λ dependent Hamiltonian H(λ) was introduced, with λ ranging from 0 to 1, H(0) = H(X) and H(1) = H(I). Since the only difference between the X state and the I state is the extra side chain atoms of residue X (except for alanine which has the same number of atoms as the I state), the computational alchemy corresponds to the annihilation of these atoms up to Cβ. A path was built to remove the side chain atom charges first followed by the van der Waals parameters elimination. The side chain charges were removed using linear λ transformation with 21 windows and λ set at 0.05n (n = 0, 1, …20). Then the van der Waals (VDW) interaction was eliminated using a soft core Lennard-Jones potential which converts the side chain atom types to dummy atoms [42], with α = 0.5, σ = 0.3, and a soft-core power of 1. These dummy atoms have the VDW radius and the depth of VDW potential well, as well as bonded parameter equal to zero. As a result, in the VDW transformation the bonded interactions with the side chain atoms (up to Cβ) of residue X were also removed. Forty-one windows were used for the VDW transformation, with λ equal to 0.025n (n = 0, 1, …40). Each window was run for 250 ps, and the last 200 ps data were used for free energy evaluation which was done using Bennett's acceptance ratio method [43]. Since X was mutated to I simultaneously in both subunits, the computed electrostatic and VDW free energies were scaled by 0.5. The total free energy change of the X→I alchemical transformation ΔG(X→I) = ΔGele(X→I)+ΔGvdw(X→I), where ΔGele(X→I), ΔGvdw(X→I) are free energy changes corresponding to Coulomb switching and VDW switching respectively. The same relationship applies for the Y→I transformation. The free energy change of the X→Y transformation, ΔG4 = ΔGele(X→Y)+ΔGvdw(X→Y) = (ΔGele(X→I)−ΔGele(Y→I))+(ΔGvdw(X→I)−ΔGvdw(Y→I)). The error for each term was estimated as follows. We divided the 200 ps simulation data to five blocks and calculated the standard deviation of corresponding ΔG values (the free energy difference between two neighboring windows). We used the standard deviation as the estimation for the error of ΔG. The typical average correlation time for ΔH (the Hamiltonian difference between two neighboring windows) is about 3 ps (Figure S4), comparable to the value from the literature [44], suggesting the data from five blocks are independent in the simulation time span. However, longer correlation time that is not sampled well in the simulation may still contribute to the error of ΔG. When comparing the computed ΔG with the experimental value, one has to bear in mind that this error can be a source of the discrepancy.
Unlike the folded state, which has a well-defined structure, the unfolded state is difficult to model. It has been proposed to use a GXG tripeptide to mimic the unfolded state where X is the residue intended for mutation. This simple model yields reasonable results compared to the experimental ΔΔG values [19] and thus was adopted in this work. Similar to the folded state, the two step transformations were used to calculate the free energies and the same MD simulation protocol was employed for the GXG model. The free energy difference ΔG3 between the unfolded X and Y mutants can be calculated with the equation similar to that for ΔG4 as described above. The folding free energy change ΔΔG(X→Y) = ΔG4−ΔG3 = ΔΔGele(X→Y)+ΔΔGvdw(X→Y) = (ΔGele(X→Y, F)−ΔGele(X→Y, U))+(ΔGvdw(X→Y, F)−ΔGvdw(X→Y, U)), where subscript F (U) stands for the folded (unfolded) state. ΔΔGele(X→Y) and ΔΔGvdw(X→Y) are the electrostatics and VDW contributions to the folding free energy differences between X and Y mutants. The error of ΔΔG(X→Y) is propagated from that of ΔG. It is worth noting that ΔΔGele(X→Y) and ΔΔGvdw(X→Y) depend on the path selected for the free energy integration thus are not uniquely defined. But the free energy decomposition provides valuable information which cannot be obtained by ΔΔG(X→Y) alone [45]–[47].
Sequence alignment analysis
Bacillus Subtilis FbFP was used as the initial target for sequence search in NCBI nonredundant protein database. Sequences were aligned using BLAST [48] and those with identity higher than 90% were excluded. A total of 83 sequences were collected and all have identity higher than 45% with Bacillus Subtilis FbFP. The frequency of one amino acid type at a specific site was defined as the total number of the occurrence of the amino acid at this site divided by 83, the total number of sequences.
Cloning, expression and purification
The DNA encoding residues 20–147 of FbFP and a His tag at the C-terminus was ligated with the vector pET-30a digested with the same restriction enzymes. The ligation mixture was transformed into an E. coli strain DH10B. The correct coding sequence of the cloned fbfp gene was verified by DNA sequencing. The expression construct (pET-30a-fbfp) was then transformed into E. coli strain BL21 (DE3). All the mutations were made by PCR-based site-directed mutagenesis and verified by DNA sequencing. All the mutants were expressed and purified in a similar way. Briefly, 250 mL of LB medium containing 50 µg/mL kanamycin was inoculated with a fresh colony of expression strain BL21 (DE3) containing pET-30a-fbfp. The culture was grown at 37°C with vigorous shaking (∼200 rpm). When the OD600 of the culture reached 0.8, the expression of YtvA LOV was induced by the addition of IPTG to a final concentration of 0.5 mM. The culture was then grown for an additional 4 h. The cells were harvested by centrifugation, washed twice with water, resuspended in 20 mL of 5 mM imidazole, 0.5 M NaCl, and 20 mMTris-HCl buffer (pH 7.9), and disintegrated by three passes through a French press. The disintegrated cells were centrifuged (9600 g, 4°C, 20 min) and the resulting supernatants were purified by Ni-NTA affinity chromatography (Novagen) to homogeneity as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined by Bradford protein assay [49].
Fluorescence measurements and data analysis
The decrease of FMN fluorescence when heating FbFP to a higher temperature allows one to monitor protein folding and unfolding by fluorescence spectroscopy (Figure S3). By setting the excitation wavelength at 450 nm (maximum absorption frequency at room temperature [24]), the maximum change of fluorescence on thermal denaturation was obtained with an emission wavelength of 506 nm (Figure S3). The final protein concentration was 4.2 µM of monomer. Spectroscopic measurements were carried out in a thermostated cuvette holder and the temperature was gradually increased. For each data point at different temperature, a delay of 10 min was applied before performing fluorescence measurement to allow the full equilibration between folded and unfolded states. The errors were estimated based on duplicated measurements. The thermal denaturation of FbFP is reversible based on the observation that heating the protein to a higher temperature (e.g. 70°C) attenuates the fluorescence by more than 80%, but it is fully recovered when the protein is cooled to room temperature. The Gibbs free energy of unfolding at a specific temperature is written as [50],(1)where is the melting temperature (half of the protein population is unfolded), ΔH is the unfolding enthalpy at . ΔCp is the difference of heat capacity in the folded and unfolded state which is assumed to be temperature independent. Ct is the total protein monomer concentration. This equation shows that the Gibbs free energy as well as the melting temperature is related to the protein concentration [50]. Thus, in this work for different mutants, the same concentration (4.2 µM) was used to eliminate this effect. The fluorescence intensity (IF) at specific temperature can be expressed as [51],(2)where αF (αU) is the fluorescence of the folded (unfolded) state at zero K, and βF (βU) is the slope of the corresponding temperature dependence. Incorporating eq. 1 to 2 shows there are a total of seven parameters (ΔH, , ΔCp, αF, αU, βF, βU) to be fitted to experimental fluorescence intensities. A simpler five-parameter model was also tested (ΔH, , ΔCp, αF, αU, βF = βU = 0). Before data fitting, fluorescence intensities were scaled so that the maximum value was equal to 100. The fitting for FbFP using seven parameters yielded a reduced χ2 of 14.7 while fitting to the five-parameter model gave χ2 of 17.9, from which an F statistic value of 1.31 was obtained. This number was smaller than the critical F(2,12) value of 3.88 (α = 0.05), indicating the complex seven-parameter model did not yield a significantly better fit. For all other mutants, the five-parameter model fittings also produced satisfactory results, and thus were selected for all fluorescence data analysis throughout this work. By adding random Gaussian distributed noise to the experimental data (with the standard deviation equal to the fluorescence measurement error), 100 synthetic data sets were created and fitted to estimate the errors of the fitting parameters. All the fittings were performed using an in-house script. The results show that ΔCp and ΔH have a considerable error derived from Gaussian noise (Table S1), but the error of ΔG is much smaller, with an average of 0.14 kcal/mol for all single mutants (Table 1). It is known that the ΔCp parameter is difficult to determine with the fitting of protein thermal denaturation fluorescence curve. Apparently the errors of ΔH and ΔCp cancel out each other. Thus, ΔG can be determined with accuracy higher than ΔH and ΔCp.
Computational screening
Starting from the crystal structure (pdb code: 2PR5), 2394 single mutants (126×19, saturation mutagenesis) were built and quickly screened by FoldX, which predicted 712 mutants from 94 sites have improved thermostability. Because it is very time consuming to do free energy calculations for all the mutants, the number of mutants was reduced to 103 using the following criteria: 1). For each site, if several mutations show improved thermostability, only the ones with relatively larger ΔΔG are selected. 2). For mutations involving charged residues, only those on surface that may form salt bridges are selected. Free energy calculations suggest that 70 of 103 mutants are more stable than the wild type (WT) FbFP, among which 40 mutants from 22 sites have ΔΔG more negative than −1 kcal/mol. The distribution of the 22 sites is shown in Figure S1.
Experimental validation
Thirteen mutants that have relatively more negative ΔΔG were tested experimentally, including V25I, T30M, A33Y, T50M, T54Y, A81M, V88L, V90I, L106M, N107F, D109E, V120I, and N124F. Melting temperatures of the wild-type (WT) and mutants were determined (see Materials and Methods) and are listed in Table 2. The WT FbFP has a of 42.8°C, indicating a moderate thermostability. The temperature fluorescence plot suggests at 37°C, 25% of fluorescence is already lost compared to that of 14°C which is the starting temperature of measurements (Figure 1). Eight of the tested mutants displayed improved stability (Table 2), whereas four mutants are less stable and one mutant L106M loses fluorescence at room temperature, presumably because L106 points to the active site and the mutant might perturb the FMN binding pocket. The success rate of 62% is encouraging, highlighting effectiveness of the computational screening. Locations of the eight thermostable sites with higher are shown in Figure 2. As can be seen, five sites are from the dimeric interface including V25, N107, D109, V120 and N124, suggesting that forging stronger interactions between subunits is an effective way to increase FbFP thermostability.
The fluorescence intensity of the bound FMN is used to monitor the protein denaturation. As can be seen, the mutants have higher percentages of fluorescence at elevated temperature than WT suggesting mutations increase FbFP thermostability.
WT residues of the mutated sites are highlighted in yellow and labeled in red. The two subunits are drawn in grey and dark cyan respectively. Residues H22, V25, N107, D109, M111, V120 and N124 are from the dimer interface. The figure was drawn based on FbFP x-ray structure 2PR5 by using Discovery Studio Visualizer program.
Among the tested mutants, V120I has the highest (54.2°C), 11.4°C higher than that of the WT. It is surprising that mutating a valine to an isoleucine, equivalent to substituting one Hβ atom with a methyl group, has such a dramatic effect. In the free energy calculation, the force field GROMOS53A6 was used where the side chain charges are zero for these two residues. So the stabilization of the methyl substitution is exclusively from the van der Waals interaction (Table 1). The analysis of V120I MD trajectory suggests that in subunit one this methyl is in contact with V27′ and I29′ while in subunit two it contacts I29′, I29 and M111 (prime denotes residues from the neighboring subunit, Figure 3A and B, table S2). The contact differences in two subunits reflect the asymmetry of the dimer.
N107F and N124F have second (50.9°C) and third (50.2°C) highest among the thirteen mutants. The free energy calculation suggests that the stabilization of the mutants is mainly from stronger van der Waals interaction (Table 1). It is worth noting that the free energy decomposition to the electrostatics and van der Waals is only an approximate process [46], [52], [53]. In this work, we decompose the free energy to two components, the free energy change of Coulomb switching and that of VDW switching, slightly different from the literature. Though the decomposition is exact, the values depend on the integration path and the process should only be considered as a qualitative analysis. However it can be linked to the structure and interaction more easily, thus provides important insight about the enthalpic part of the free energy. The MD simulation of the WT shows that N124 from subunit 1 is in contact with R24′ and V25, whereas N124 from subunit two is in contact with V23′, V25 and E105. In the mutant N124F, F124 forms much more contacts than WT N124 in both subunits (Figure 3C and D, Table S2). But for N107F, the difference is less dramatic. Free energy calculations indicate van der Waals contributes more than the electrostatics to ΔΔGs for all stabilizing mutants except D109E, revealing the importance of enhancing core packing to protein stability. The carboxyl group of D109 forms a hydrogen bond as an acceptor with the phenol group of Y141′. Mutating D109 to a glutamic acid extends the side chain and an extra hydrogen bond is formed with the N107 side chain amide (Table S3).
Rational design of more mutants
The high success rate of the stable interface mutants prompts us to search for more candidates in the region. Most of the discovered stabilizing mutants including V25I, N107F, D109E, V120I and N124F are from the middle of the beta-strands that form the interface. In searching for other stabilizing mutants, more effort was put in interface regions away from the middle part because the combination of mutants close to each other may not be additive. Residues H22, M111 were identified. H22 was mutated to a lysine to promote a potential salt bridge formation with E105′ or E133′, or to a tryptophan to form more contacts. M111 was mutated to a phenylalanine to increase this residue's contacts with the neighboring subunit. Free energy calculations demonstrate that all three mutants have negative ΔΔG values, suggesting they are more stable than the WT. Inspection of the mutants N107F and N124F MD snapshots implies it is possible to further stabilize the protein by mutating F107 or F124 to a tyrosine to form more favorable electrostatic interactions with the surroundings. This observation is supported by the free energy calculations that N107Y (N124Y) has a more negative ΔΔG value than N107F (N124F) and the difference is mainly from electrostatic interactions (Table 1).
Five mutants H22K, H22W, M111F, N107Y and N124Y were tested experimentally and the corresponding values were listed on Table 2. All five mutants were proved to be more stable, consistent with the computational results, among which N124Y has the highest (63.8°C), 21.0°C higher than that of the WT. The analysis of the N124Y MD trajectory suggests besides close contacts with various residues (Table S2), N124Y forms extra hydrogen bonds. Specifically, in subunit one the side chain hydroxyl group of N124Y is h-bonded to V23′ and V25′, and in subunit two it is h-bonded to V23′ and Q129 (Table S3). These hydrogen bonds are expected to contribute the most to the 13.6°C increase of the F to Y mutation. In comparison, N107Y is also more stable than N107F but the difference is much smaller, only ∼1°C. M111F has a of 56.5°C, 13.7°C higher than the WT. MD simulation suggests F111 in subunit one forms close contacts with I29, I29′, Y41′, Y118 and V120, and in subunit two this residue is in contact with I29′, V40′, Y41′, Y118 and V120 (Figure 3G and H,table S2). H22K and H22W have values 3°C and 7°C higher than the WT respectively. MD trajectory shows K22 forms salt bridges (Table S4), and W22 forms several contacts with the surroundings (Table S2).
Multiple mutations combination
About a dozen mutants have higher than that of the WT, providing an opportunity to improve the thermostability of the protein further by combining multiple mutations. But there are many ways of combining them. Two factors were considered, the Δ (melting temperature difference with the WT) and the distance between different mutants. Several multiple mutants were tested, among which double mutant N107Y-N124Y displays better thermostability than both N107Y and N124Y, and triple mutant N107Y-N124Y-M111F has its higher than all three single mutants (Table 2). However, not all the multiple mutants have higher thermostability than the single or double mutant. For example, N107Y-N124Y-H22W has a value 2°C lower than that of N107Y-N124Y, though H22W has a 7°C higher than that of the WT. Similarly, N107Y mutation increases the by 9°C, but N107Y-V120I is only marginally more stable than V120I (Table 2). So the additivity of the thermostability seemly holds true only for certain multiple mutants. N107Y-N124Y-M111F is the mutant with the highest presented in this work, which is 31°C higher than that of the WT. To ensure that the triple mutations do not compromise the fluorescence brightness of the protein, its fluorescence quantum yield was determined, QF = 0.38, comparable to that of the WT FbFP 0.39 [22].
Performance of the computational methods
FoldX method provides a quick way to evaluate the mutational effect on the protein stability. It takes about 82 hours to screen the amino acids at each site for FbFP (∼2400 mutations) in a dual processor (2.9 GHz Intel) computer. In this work, FoldX predicts the thermostability of twelve out of seventeen mutants correctly, suggesting that overall FoldX is effective in identifying stable mutants. Three mutants, T50M, A81M and V88L, which are less stable than WT, were not predicted correctly. Furthermore, H22W and M111F were predicted less stable than the WT with the ΔΔG of 0.40 and 4.25 kcal/mol respectively but the experimental ΔΔGs are −1.16 and −2.87 kcal/mol. Further inspection suggests that steric clashes exist for the M111F mutant with its surroundings. By using the last MD snapshot of the 1 ns NPT M111F simulation as the FoldX input, a ΔΔG of −0.70 kcal/mol was obtained indicating M111F is more stable. For mutations causing steric clashes, it has been recommended to either soften the VDW repulsion [16] or relax the backbone atoms slightly [15] which is not allowed in FoldX. The correlation between the FoldX predicted and experimental ΔΔG is shown in Figure 4A. A Pearson correlation coefficient RP of 0.19 was obtained, suggesting that FoldX only provides qualitative information of the relative thermostability. For mutations at the same site, e.g. N107Y/F or N124Y/F, FoldX also predicts the relative thermostability in the wrong trend. For example, N124Y is significantly more stable than N124F but FoldX predicts ΔΔG of −2.68 kcal/mol for the former and −3.67 kcal/mol for the later.
The best fit line is ΔΔG_com = 0.233ΔΔG_exp −0.72 kcal/mol in panel A, and ΔΔG_com = 1.38ΔΔG_exp −1.42 kcal/mol in panel B.
Free energy calculations, derived from statistical mechanics, offer a more rigorous way to estimate mutational effect on thermostability but with a much slower speed. A single point mutation of FbFP needs 440 processor (AMD 1.9 GHz) hours, about 13,000 times slower than the FoldX approach. Thirteen of seventeen mutants were predicted with the correct sign of ΔΔG, except T50M, A81M, V88L and V90I. The predicted and experimental ΔΔGs are shown in Figure 4B. Two mutants, T30M and D109E, are apparent outliers. Excluding these two, a correlation coefficient RP of 0.68 was obtained with the best fitted line of ΔΔG (predicted) = 0.59ΔΔG (experimental) −1.80 kcal/mol. A negative intercept suggests that free energy calculations systematically overestimate the stability of the mutants, which may be caused by the force field error. Excluding the two outliers T30M and D109E, the mean absolute error is to 1.7 kcal/mol. Applying the linear correction reduces the error to 0.93 kcal/mol, comparable to results in the literature [19]. Compared to FoldX, the free energy method is more accurate and yields semiquantitative results. But there is still room for improvement.
There are a total of 13 stabilizing mutants from 10 sites (Table 2). It is interesting to see that 7 of the 10 sites are from the dimeric interface, except T30, A33 and T54. Unlike the interface, mutants around these three residues have not been thoroughly examined. In fact, about 2/3 of the 40 stabilizing mutants predicted by the free energy method (Figure S1) are not from the interface region among which about half of the mutation sites have not been tested experimentally. More effort will be spent to search for single mutants away from the dimeric interface and tune computational methods for screening multiple mutants.
Consensus analysis
A multiple sequence alignment was carried out for a total of 83 FbFP sequences from the NCBI protein database. It is striking that none of the 13 stable mutants discovered in this work have the highest occurrence frequency (defined as the probability to have a specific amino acid at a particular site) at the mutated site (Table S5). For example, for residue 120, valine has the highest occurrence frequency of 78.3% which is much larger than that of isoleucine 15.7%, but V120I improves the thermostability by 11.4°C. In fact the majority of the stable mutants have the occurrence frequency at mutated sites smaller than that of the WT. This finding strongly suggests that FoldX and free energy calculations explore very different sequence spaces from the consensus analysis method.
In this work, we developed a protocol to engineer a more thermostable FbFP. Firstly, FoldX followed by free energy calculations are performed to identify stable single mutant candidates. Secondly, mutants predicted with more negative ΔΔGs are selected for the experimental validation. For FbFP, the majority of stable mutants are from the dimeric interface which is a "hot spot" of the protein. Thirdly, more single mutants from the "hot spot" are selected and verified by free energy calculations first and those with more negative ΔΔGs are subject to experimental tests. Finally, stable single mutants are combined to further improve the protein thermostability. A flow chart of the protocol is shown in Figure 5. Thirteen out of eighteen tested FbFP single mutants are more stable than the WT. Most of the stabilizing mutants form a better core packing. The best triple mutant N107Y-N124Y-M111F increases the by 31°C without compromising the fluorescence quantum yield. The experimental test is by no means extensive but the effectiveness of the protocol has been demonstrated. The combined experimental and computational method adopted in our work adds a new tool to enhance protein thermostability, which can be extremely useful when conventional methods (e.g. directed evolution) fail.
Briefly, FoldX followed by FEC (free energy calculation) are used to search for potential thermostable single mutants, from which a dozen are selected for experimental tests. The distribution of thermostable mutants is analyzed to identify the "hot spot". Then more mutants in the "hot spot" are calculated by FEC and those predicted to be more stable are tested by experiments. Finally all stabilizing mutants are pooled together and multiple mutants are combined to further improve the protein's stability.
Supporting Information
Figure S1.
Distribution of the thermostable mutations in the 3D X-ray FbFP structure (pdb code: 2PR5). The 40 mutants are: V23I/M/W/K, V25I, T30L/M, A33Y/L, V40I, T50M/I, T54L/Y, V75I/M, A81M, V88L/Y, V90I, N94M/Y, L106M, N107M/F, D109E, T117L, V120I/L/M, G121M/Q, N124I/M/F, Q129M, S139M and T141M. Residues highlighted in yellow are those with ΔΔG more negative than −1 kcal/mol. The stability was predicted by free energy calculations.
Figure S2.
A). Thermodynamic cycle built for free energy calculations. B). The intermediate state I designed to connect the X and Y mutants.
Figure S3.
Fluorescence emission spectra of FbFP at different temperatures. The excitation wavelength was set at 450 nm.
Figure S4.
Correlation time τ of ΔH (the Hamiltonian difference between two neighboring windows) as a function of λ in the F111 electrostatic (panel A) and vdw (panel B) transformation. The average correlation time is 3.0 ps for the electrostatic transformation and 2.5 ps for the vdw transformation.
Table S1.
ΔH, , ΔCp, αF, αU values derived from the fluorescence curve fitting for FbFP WT and mutants.
Table S2.
Contacts of selected residue side chains in the WT and mutants.
Table S3.
Hydrogen bond lists of selected residues.
Table S4.
Salt bridges formed with K22 in H22K mutant.
Table S5.
Frequency of a specific amino acid occurrence at selective sites of the WT and mutants from the alignment of 83 FbFP sequences.
The authors would like to thank Shanghai Supercomputer Center for providing computer resources.
Author Contributions
Conceived and designed the experiments: LY. Performed the experiments: XS YW ZS JH TL. Analyzed the data: XS YW. Contributed reagents/materials/analysis tools: ZS JH TL. Wrote the paper: XS LY.
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Thu, 02 Mar – Fri, 03 Mar (1 Night) | 1 Room | 1 Guest
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text: When ds1 was 7 months I went back to work part time and being able to sit in a coffee shop over my lunch hour reading a book was heaven!! Now I've been a sahm for a year and a half with 2 little ones and the only down time j get is when they are in bed. My dh would take them out if I asked but he's not great at thinking ahead and offering so I just get into the habit of not bothering. Stupidly if I do get some alone time to go to dr appointment for example I end up rushing back as I feel guilty that I've left dh on his own with the 2 of them. Crazy I know as he's always fine but it means I dont actually enjoy the alone time. Can't wait to be in a position to go back to work and get some of the real me back.


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text: Petition calls for B.C. Family Day date change to align with other provinces'Unite Family Day' movement supported by Green, NDP parties02/13/2017|payroll-reporter.com|Last Updated: 02/14/2017A child shovels snow to make a small hockey rink on the frozen Trout Lake during a spell of cold weather in Vancouver, Jan. 7. REUTERS/Chris Helgren VANCOUVER (CP) — As British Columbians enjoy the long weekend, thousands say the province is celebrating Family Day at the wrong time.A petition on Change.org is calling for B.C. to align its Family Day holiday with six other provinces, including Alberta and Ontario, which hold it a week later on the third Monday of February.The petition argues that having the holiday on the second Monday of the month makes it redundant for many workers who still have to do business with parts of the country that don't have the day off.The call to "Unite Family Day" is supported by B.C.'s Green party leader Andrew Weaver, New Democrat MLA David Eby and a number of investment professionals.B.C.'s Family Day has been in place since 2013 after public consultations that favoured the earlier weekend for better skiing conditions and reduced traffic over the border into the U.S., which celebrates President's Day the following week.As of Monday morning, the petition had over 18,000 signatures. To Read the Full Story, Subscribe or Sign In Remember Me Forgot Password If you are a current Subscriber, please click here to set-up or update your login information. Add Comment * Title * Name * Email Anonymous * Comment


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Kia appoints Audi TT designer to shake up new range
Kia has some grand designs ... both in terms of the tin it hopes to sell and its ambitions to be much higher up the motoring food chain.
Now, one of the world's leading designers, Peter Schreyer, has been hauled in too shake up the future shape and styling of Kia's regenerated range. Noted for his work on the Audi TT (and, unfortunately, the new Beetle), Schreyer will oversee the implementation of the company's new designs, which it is hoped will bring the Korean also-ran more into the hearts, minds, pants and wallets of the younger, more adventurous car buyer.
With the Picanto and Sedona doing fairly well and the new Sorento a massive improvement on the old one, Kia has seen a boost in its sales of late as it fights hard for the budget end of the market.
Euisun Chung, President and CEO of Kia Motors Corporation, said: "As we continue to develop the Kia brand across all key regional markets, we must continually enhance our range of vehicles through world class, innovative advances in product design. This appointment underlines our commitment to meeting and exceeding customer expectations by accurately reflecting modern aesthetics and regional and cultural sensibilities".
"We are focused on boosting our competitiveness on the world stage by dramatically enhancing the design element of our vehicle lineup while adding a strong emotional dimension to the brand."
Schreyer will control Kia's regional design centres in Korea, Germany, Japan and the US, and he is slated to make his first public address at the Paris Motor Show in September. Kia's first ever vehicle designed specifically for European customers - a five door family hatchback codenamed ED - also make its world premiere at the show.
A small word of warning, though. When legendary designer Giorgetto Giugiaro (of BMW M1, FiatPanda, VW Golf and Maseratti 3200GT fame) went to Daewoo, he gave us the Matiz, Kalos and Lacetti. So that worked out well, then...
Jeep launches Renegade Desert Hawk SUV, limited to 100 models in the UK
Range Rover Velar designed to fill the void between the Evoque and Sport
Smart Electric Drive (2017) first drive: Nippy inner-city transport sprouts...
BMW 5-Series (2017) review: Saloon car perfection?
- Jeep launches Renegade Desert Hawk SUV, limited to 100 models in the UK
- Range Rover Velar designed to fill the void between the Evoque and Sport
- Smart Electric Drive (2017) first drive: Nippy inner-city transport sprouts a plug socket
- BMW 5-Series (2017) review: Saloon car perfection?
- Jaguar drivers in UK can now pay for Shell gas without leaving their car
- Honda Civic 2017 first drive: Classic hatch gets a millennial makeover
- Jaguar F-Pace review: Outpacing its SUV rivals?
- Morgan made a £6,660 kid version of its all-electric EV3 vehicle
- Audi MMI explored: A deep dive into Audi's in-car infotainment systems
- Audi A5 (2017) review: Sporty looks, refined drive
- Mobile World Congress 2017: Nokia, LG, Samsung, Huawei, Sony smartphones and more
- Motorola Moto G5 Plus vs Moto G4 Plus: What's the difference?
- Best of MWC 2017: Samsung, LG, Sony, Huawei and more
- Sony Xperia XZ Premium preview: The complete Xperia package
- HTC Ocean leak suggests phone will offer 'Edge Sense' display feature
- Huawei P10 preview: The lean, green photo machine
- Sony Xperia XZs vs XZ vs Xperia Z5: What's the difference?
- Porsche Design Book One is a ridiculously plush 2-in-1 Windows dream device
- Huawei P10 and P10 Plus: Release date, price, specs and everything you need to know
- LG G6 preview: All screen, no frame


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Christian Dad regrets tweet at 'Supergirl' to 'tone down homosexual messages'Friday, January 27, 2017"I was a bit narrow-minded in my choice of words... not understanding my choice of words could be taken as offensive."
Christian radio host's daughter comes out as bisexual in powerful letterSaturday, January 14, 2017Her father, Rick Burgess, is cohost of syndicated right wing evangelical talk show Rick and Bubba.
Lesbian couple named co-pastors of D.C.'s Calvary Baptist ChurchThursday, January 12, 2017This isn't the first Baptist church to hire an openly gay minister or even to have lesbian co-pastors, but it is still known for making history.
RNC appears to compare Trump with Christ then denies it and blames the mediaMonday, December 26, 2016The RNC Christmas message included language many felt compared Trump with Jesus, but the incoming White House Press Secretary says that's nonsense.
Nativity ornaments replacing Mary and Joseph with same-sex parents spark hateWednesday, December 21, 2016Despite the outcry by conservative Christians, the artist behind the nativity scenes is once again selling the ornaments.
Antigay forces launch new international hate groupMonday, December 12, 2016The International Organization for the Family won't just fight same-sex marriage but reportedly will challenge LGBTQ rights around the world.
Antigay preacher to mall kids: 'Santa is not real'Monday, December 12, 2016Evangelical pastor David Grisham recorded himself trying to "teach" kids "that Christmas is about Jesus Christ."
Tens of thousands in Trump's new friend Taiwan march against marriage equalityTuesday, December 6, 2016"We oppose homosexual marriage... because the family system comprised of marriage between one man and one woman is the foundation of society."
Pastor of HGTV's 'Fixer Upper' hosts says their antigay church is not antigayFriday, December 2, 2016"We are not only not antigay, but we are pro-helping people in their journey to find out who God is and who He has made them to be."
Russian Orthodox patriarch compares same-sex marriage to Nazi laws and apartheidWednesday, November 30, 2016"We believe this new trend poses a significant threat for the existence of the human race."
Page12345...Continue Reading »


text: Feather paisley maxidressItem A7536Size:Size Charts Quantity:123456789Add to Bag Wishlistshare:size & fit Fitted at waist.58" from high point of shoulder (based off size 6).Product Details It's not officially summer until you break out the maxi. In cotton and silk and finished with a flattering drawstring waist, this dress is incredibly easy to wear. Don't be surprised if you start to neglect the rest of your wardrobe.Cotton/silk.Back zip.Lined.Machine wash.Import.Online only.Item A7536.


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Video Recipes
"Pachakam" means cooking in kerala and Pachakam.Com welcomes you to the magnificent world of easy and healthy Indian cooking with a probe into the fascinating Indian cooking recipes. Cooking is not just about making food but is an art of presenting your culinary skills to those who love food with a different flavor and Indian cooking, by no means is an exception.
healthy curry for breakfast
Category :
Published On: 03 Nov 2013
Heat the oil in a pressure cooker and add cumin seeds, minced garlic, ginger and green chillies.
Fry till the spluttering stops and add the chana daal, chopped vegetables, and all the dry masala powders and salt. Cook for 3-4 minutes. At this stage, there should not be too much water in the bhaaji.
Add the chopped sua/dill, spinach/palak and khatta bhaji or tomatoes to this and mix well.
Add 3-4 cups water and pressure cook for 7-8 whistles.
Open the lid of the cooker and mash the sai bhaji with a ravi/hand churner/kavvam (traditional wooden or stainless steel churner) rolling it back and forth between your palms or you can use a hand blender.
Adjust the consistency of the sai bhaji by adding water or by drying up excess water. The bhaji should be thick and semi-dry.
Serve with steamed rice or roti.
Any vegetable like french beans, carrots, brinjal can be added to the sai bhaji.
its a traditional kerala fish curry with Mullan Fish....
Easy to make chicken biriyani...
The oyster mushroom, is a common edible mushroom. there is ...
Punjabi style pumpkin curry is very famous dish with sweet a...
Radish offers many health and nutritional benefits. Radishes...
Raw banana coconut stir fry is a simple and healthy stir fri...
Nizana Shammas is a hosewife who has lots of interest n cooking. She is an expert in providing various traditional recipes.
Renukadevi Kumanan is a well versed person in providing varities of South Indian recipes and also some modern recipes. She used to try varities of recipes from different cuisines.
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This is the full research report for 22.214.171.124, which is an IP address.
Administered by ARIN
Argon Data Communication
Corporate / Direct Member IDNIC
Jl. Siti Jenab No. 79, Cianjur, Jawa Barat 43211
Afriyandi Afriyandi
Jl. Siti Jenab No. 79
Cianjur, Jawa Barat 43211, Jl. Siti Jenab No. 79
Telephone: 6281320515161
Fax: 62263272658
Afriyandi Afriyandi
Jl. Siti Jenab No. 79
Cianjur, Jawa Barat 43211, Jl. Siti Jenab No. 79
Telephone: 6281320515161
Fax: 62263272658
22.214.171.124 is the IP address you have a ran a report for on February, 23, 2017.
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Makeup by Renette
- Face Painter in Orlando, FL
- Travels up to 200 miles
- $100-$300
- Plan Your Event
About Makeup by Renette
With over 12 years experience, Makeup by Renette Inc. is a fully insured company that offers high quality face painting that takes less than 5 minutes to create and are always a hit for any occasion! We only use FDA approved non-toxic makeup that washes off easily with soap and water.
Makeup by Renette has been trained by some of the best artists in the industry.
We also offer Airbrush tattoos for longer wear. They are non-toxic as well and last up to 2-4 days.
Balloon creatures are a great way to add fun to any party. Choose from a variety of animals, swords and fun to wear hats!
Book us for your next event!
Wow! Phenomal!
By Patrick A. on March 20, 2016
We attended an Easter event with several other vendors. The other vendors were good, but Renette was the best. My daughter is rather shy around strangers. Renette had her so comfortable that in no time my daughter was telling her what she wanted. Renette was not only a good listener, and artist, she intuitively used colors that matched my daughter's outfit. She is a gifted young artist.
|Hired as:
|Children's Party Entertainment, Face Painter
Response from Makeup by Renette:
Thank you for the wonderful review! Im glad your daughter enjoyed her face paint:)
Two for two!
By firstname.lastname@example.com on July 28, 2013
We've always been impressed by Renette's artwork, but within the last year, we booked her for two different parties and our make-up was fantastic both times. We look forward for the next party for an excuse to have her do more impressive work. :)
|Hired as:
|Face Painter
Response from Makeup by Renette:
Thank you so much, David and Patrick! I always enjoy trying new makeup ideas! Thank you again and I look forward to more fun designs!
superhero party
By Lisa W. on July 27, 2013
Renette was a great vendor!! she was on time, professional and really great with kids and adults!! Will definitely work with her again!
|Hired as:
|Face Painter
Response from Makeup by Renette:
Thank you Lisa! your party was a lot of fun! thank you for the great review and I look forward to working with you again soon!
Face Painting Birthday Event Review
By Sarah M. A. on April 10, 2013
Makeup by Renette was referred to me by a friend and my expectations were exceeded. The artistic capabilities of the artist were amazing, she was quick and really worked well with the kids, they had a great time. She was prompt, and had great communication in the entire event planning. I highly recommend Makeup by Renette and look forward to using this company in the future.
|Hired as:
|Face Painter
Birthday party for grandson
By Betty P. on April 9, 2013
Renette arrived early to our Bonnet Creek Resort unit and stayed the promised length of time. Arriving early was good as it gave my grandson Connor time to see what she was there for before the guests arrived. She immediately gained his confidence so that he readily agreed to be the first to have his face painted. She proceeded to decorate the faces of 5 children and 10 adults in a very few minutes. It was great fun and put all in a very happy birthday party mood!
|Hired as:
|Face Painter
Response from Makeup by Renette:
Thank you for the great review, Betty! It was a pleasure painting you and your family for Connor's special day!
Highly recommend!!!!
By Melanie T. on March 12, 2012
I recently had a birthday party for my 12 year old and some of her friends. I hired Renette to help make the party more memorable for the kids and I have to say I was very pleased with everything. I actually had her do makeup and balloons for the kids ranging from 6-41 (yes, I had my face done too)) and I was extremly pleased. So much so that I actually recommended her to the facility where I had the party and they are going to recommend her from now on also. She was great with the kids and did an amazing job with the kids requests for various styles and colors. Even the adults who were present were very impressed. I would highly recommend her and will surely use her again!!!
|Hired as:
|Face Painter, Balloon Twister
Response from Makeup by Renette:
Thank you Melanie!
It was a pleasure painting at your daughter's birthday. I had a great time and thank you again:)
Renette is the best!
By Fameisha G. on January 29, 2012
We had Renette as our facepainter and balloon artist at my daughter's 5th birthday party (1/28/12) and she was awesome! Her artistic ability and friendliness, made her the hightlight of the party. She was on time and very professional. We had a hello kitty themed party, so she made my daughter look just like hello kitty (which made her so happy!). We probably had about 15+ kids and 25+ adults and Renette made them all feel special. Some adults were even brave enough to have their faces painted (even me)! Everyone had a blast. We would definitely book her for another party! I've already started to give her contact info to all the mom's at the party (who all adored her!) Great Job Renette :-)
(Nemya's mom)
|Hired as:
|Face Painter, Balloon Twister, Children's Party Entertainment
Review by Teri F.
By Teri F. on March 26, 2011
Renette did an amazing job face-painting at my son's 7th birthday party. Kids who initially were hesitant to get their faces painted got over their fears and jumped into her chair as they saw her working with the other boys and girls (and some of us adults, too!). All of the kids and parents were thrilled with her work, one of them even stating that although her business card states that she does most work in under 5 minutes, that she could actually change it to even faster than that! Her work is fast, but it is of the highest quality. Every parent left with her business card at their own request!
|Hired as:
|Face Painter
Review by Patricia L.
By Patricia L. on November 29, 2010
This was my first official party I planned for my son's first birthday and it was a HIT!!! Almost everyone at the party got their face painted and it all turned out so awesome! She did a lot of requests and everyone loved their face! I would definitely hire her again. Her price was worth every penny and she is very enthusiastic and personable.
|Hired as:
|Face Painter
Review by Yamile B.
By Yamile B. on May 4, 2010
Renette did a great job with our kids birthday party. We had a superhero theme party and the kids enjoyed all of the options she had. She was great with the kids. It shows that she loves what she does! Thank you, thank you, thank you Renette!
|Hired as:
|Face Painter, Superhero Party
Review by Amy R.
By Amy R. on April 28, 2010
Renette is an incredible artist! We had approximately 56 kids and she managed to paint some amazing designs on all of the kids within the two hour time period we had a lotted. She paints the kids so fast and they are absolutely beautiful. We've used her for a few years now and will definately hire her again. She was even able to paint a cute puppy dog face on my 2 year old who never holds still. She definatley has a great repoor with kids.
|Hired as:
|Face Painter
Review by Tracy F.
By Tracy F. on April 18, 2010
Renette was fantastic to work with. She was always prompt at responding to emails and phone calls when trying to book her for my son's birthday party. She was great with the kids and had a variety of faces to choose from. Renette did an amazing job with the face painting as well as adapting to all the kid's concerns and needs. She was very creative and artistic! I would recommend her for all of your face painting needs!
|Hired as:
|Face Painter
Booking Info
- Typical Gig: 60 - 180 minutes
- Fee: $100-$300
- Insurance: accidental
Additional Booking Notes
well lit area with cocktail table
Renette has been face painting since 1999. She has worked in most of Orlando's major theme parks including Universal Orlando, Walt Disney World and Sea World of Orlando.
She has a degree in Visual Arts and has experience with watercolours, acrylics, sculpting and throwing clay and even fabric design.
She currently works for Universal Orlando's makeup/prosthetics team and area resorts.
She has also had the opportunity to travel to Hong Kong for the past five years to airbrush makeup and apply prosthetics for Ocean Park's Halloween event.
Artists that have worked with Makeup by Renette Inc. are theme park trained and very professional.
The team providing the talent:
Renette Hartman
Owner, Artist
Noah Hartman
Stilt Walker, Balloon Twister
Emilie Watson
face painter
Justin davis
face painter
Influences & Inspiration
Wolf Brothers, Face and Body Art convention
Setup Requirements
cocktail table and chair
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text: Pinch to zoom Girl's Striped Pyjamas 11-14yrs by PJ Pan £44$75.51€52.73$56.38 Favourite Favourite View your favourites sizesize (required)11-12yrs13-14yrs sold out (+ £2)size (required)11-12yrs13-14yrs sold out (+ $3.43)size (required)11-12yrs13-14yrs sold out (+ €2.39)size (required)11-12yrs13-14yrs sold out (+ $2.56) quantity personalise & pay by PJ Pan Location: Perthshire, GB read seller's profile see seller's complete range more items from this seller Men's Paisley Pyjamas by PJ Pan £145$248.84€173.78$185.80 Pretty Pink Gingham Cotton Ladies Pyjamas by PJ Pan £95$163.03€113.85$121.73 Men's Dark Blue And White Stripe Pyjamas by PJ Pan £95$163.03€113.85$121.73 Men's Striped Pyjama Bottoms by PJ Pan £49$84.09€58.72$62.78 Women's Pink And Grey Check Pj Bottoms by PJ Pan £42$72.08€50.33$53.81 Women's Brushed Cotton Purple Pyjama Bottoms by PJ Pan £42$72.08€50.33$53.81 view all you may also like PRODUCT DETAILS DELIVERY RETURNS Made in Britain ask seller a question save for later Girl's pyjamas in a fun turquoise and white stripe. 100% finest cotton. Gently tapered jacket and finished with stylish turn ups and real shell buttons. Practical trousers with elasticated waist and functional drawstring. Matching ladies pyjamas available. The sizing for our children's pyjamas and pyjama bottoms are based on dual sizes, so they are designed to fit the age at the upper end of the size. The leg lengths are generous but can of course be taken up if needed. All trousers have a functional drawstring so they can be tightened if the child has a small waist. Please refer to the sizing chart below for more details (age, height, waist, chests, outside leg of trousers): 11-12 years 152cm 65cm 78cm 91cm 13-14 years 162cm 72cm 84cm 99cm made from: Fabric: Finest quality long staple combed cotton. Care advice: 40 degrees wash, easy iron fabric. product code: 180622 This product is dispatched to you by PJ Pan. You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. PJ Pan's return policy Please see notonthehighstreet.com's returns policy.