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Dee Browne – Clinic of Well-being
Dee Browne, C.H.Dhyp/C.H.Dplreg
Owner of Clinic of well-being in Lincoln.
Registered Clinical Hypnotherapist
Reiki Master & Teacher
Spiritual reader, therapist and teacher.
Psychic Medium and Clairvoyant.
Alternative and complimentary therapist
Past Life Regression therapist
Hypnothin Weight Management Consultant.
Member of the Spiritual Workers Association, the Reiki Association and the General Hypnotherapy Standards Council.
Under courses and events I have listed the next available w/shops for each subject I teach. Please note that I run regular courses all year so if the one's listed are not on suitable dates please check the website for a full list of w/shops available. There may also be the odd cancellation slot available on a earlier w/shop.
Tarot Reading/ Mediumship/ Spiritual Healing/Spiritual Development Teaching W/shops/Meditation/ Past Life & Age Regression/ Crystal Healing/ Reiki & Tarot W/shops/ Clinical Hypnotherapy/ Weight Loss/ Smoking/ Stress/ Fears & Phobias/ Confidence and much more.
Contact Dee on 07955267143
Opening Times
Fri 10am - 6pm
Sat: Teaching w/shops only
Sun: Teaching w/shops only
Location Information
Nettleham road end of Broadway on left just around first bend.
1 Review/Comment on "Dee Browne – Clinic of Well-being"


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Turkey Picadillo Chilles Rellenos
Sale price
Regular price
Since Cooked was founded in a small 500sq ft kitchen back in 2014 Chilles Rellenos, or Mexican stuffed peppers, have been one of our top sellers every time they appear on the menu. In this Whole30 friendly version of the classic dish two poblano peppers are roasted and peeled and are then stuffed with a ground turkey-potato-raisin hash. The stuffing also includes other seasonal vegetables, such as carrots, celery, and cilantro. The whole dish has hints of warming spices, such as cloves, cinnamon, and raisins though it is free of added sweeteners. It comes with a delicious tomato-cinnamon sauce and is topped with slivered almonds for extra crunch and extra nutrients.
Calories: 386
Proteins: 19g
Fats: 18g
Carbs: 37g
Proteins: 19g
Fats: 18g
Carbs: 37g

subset: commoncrawl


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Liverpool FC: The Anfield Stadium Experience for Two 11863033
Treat yourself and a fellow Liverpool fanatic to an unforgettable day at the club's iconic homeground. An exclusive tour will treat you both to the full match day experience, from the new Press Rooms and Dressing Rooms through to the Player's Tunnel and Dugout, before you hear from living Liverpool Legends about their experiences of wearing the iconic jersey during an exclusive audience and lively Q&A session. Following a delicious three course lunch in the hospitality lounges you will then pay a memorable visit to the club's interactive museum, The Liverpool FC Story, where you will find their latest and greatest exhibit, and The Steven Gerrard Collection, showcasing historic memorabilia from the Kop's favourite son. This really is the perfect experience to share with friends or family – as the club anthem goes, 'You'll never walk alone'!
- A free flow tour featuring the new Press Rooms, Dressing Rooms, Player's Tunnel and Manager's Dugout, with a multimedia handset
- A 3-course lunch served in one of the Main Stand's hospitality lounges overlooking the Anfield pitch
- A complimentary beer, wine or soft drink with lunch (1 per person)
- Exclusive photo with the LFC Legend(s) to take away with you on the day as a special gift
- MC to introduce your Legend(s) and invite them to share their experiences and personal memories of playing for the club
- An audience with your Legend(s) and a chance to ask them questions during the Q&A session
- A visit to interactive museum The Liverpool FC Story and its latest exhibition The Steven Gerrard Collection
- Opportunities for autographs and souvenir photos
- Your voucher is valid for ten months for two people
- This experience is available week round
- Any guests under the age of 18 years must be accompanied by an adult
- The experience is not recommended for those under 5 years
- All dates are subject to availability
- Legends include Phil Thompson, John Aldridge and Terry McDermott
17 Locations


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Just Landed! Check out our new direct-to-consumer range.
Compatible with all Maclaren strollers and all umbrella fold stroller brands.
Whether you're going on a day trip or a vacation these essential accessories will help you not only survive the journey, but do it in style. The Starter Kit has three accessories, including Universal Organiser in black, Universal Seat Liner in Union Jack Princess Blue print and a Cupholder in silver- the perfect combo to get you on your way. Let us help you pack!


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Religion: Judaism - General eBooks
Judaism is the religion, philosophy, and way of life of the Jewish people. Originating in the Hebrew Bible and explored in later texts such as the Talmud, Judaism is considered by religious Jews to be the expression of the covenantal relationship God developed with the Children of Israel. Rabbinic Judaism holds that God revealed his laws and commandments to Moses on Mount Sinai in the form of both the Written and Oral Torah.
Judaism claims a historical continuity spanning more than 3,000 years. It is one of the oldest monotheistic religions, and the oldest to survive into the present day. Judaism's texts, traditions and values strongly influenced later Abrahamic religions, including Christianity and Islam.
Jews are an ethnoreligious group and include those born Jewish and converts to Judaism. In 2010, the world Jewish population was estimated at 13.4 million, or roughly 0.2% of the total world population. eBookMall is an excellent source of eBooks on all aspects of Judaism.
Use our eBook search to find a specific book or author.
Top 10 Religion
- Something More: Excavating Your Authentic Self
- Seeing the Psalter
- 19 NINETEEN: God's Signature in Nature and Scripture
- Understanding Your Place in God's Kingdom
- Living Proof: Sharing the Gospel Naturally
- 128 Greatest Stories from the Bible
- David
- The Moody Handbook of Preaching
- The Power of Knowing God
- Flying Star Feng Shui


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Ireland is known for great hospitality and the fees here have helped students like me to be able to study abroad. Under the Global Peace Index, Ireland is in the top 10 safest countries and has great landscapes, gorgeous lakes and oceans and not forgetting its history– it is just mind blowing!
Cork Institute of Technology
Bachelor Business in Hospitality Management
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They also seized a loaded gun, ammunition and several knives.
CANTON Four people were arrested when police found them in a stolen car with a loaded gun, ammunition, knives and several types of illegal drugs that police believe were for sale, Stark County Jail records show.
The arrest occurred around 4:20 a.m. Sunday in the 1100 block of Pulley Place SE.
Ryan J. Tapia, 30, of 321 Thorne Ave. NE in Massillon, was driving the stolen car, jail records show.
Stark County court records show Tapia had 18 grams of crystal methamphetamine, a bag of marijuana, a bag of crack cocaine, an undisclosed amount of powder cocaine, ecstasy and several containers of hashish.
Lt. Dennis Garren, police spokesman, said officers were investigating a report of a stolen black Chevrolet Silverado pickup when they approached a group of people standing around the truck and a Toyota. They found the gun, a glass pipe and several knifes in the Toyota and the drugs in the stolen truck, he said.
Jail records show that Kevin E. Lowe, 42, of 223 E. Liberty St. in East Canton, had a loaded gun, ammunition and "several edged weapons." Police say he also tried to hide a glass pipe once they placed him in the back of their cruiser.
Samantha N. Cutlip, 37, of 4840 Clardell Ave. SW in Canton Township, had a crack pipe and an unlabeled prescription bottle containing prescription pills, the jail records show.
Brian J. Walters, 32, of 66 Rolling Acres Cir. E in Massillon, had a small amount of crystal meth and a needle, the jail records show.
Police also found a loaded magazine for the firearm in the truck, according to the jail records.
All four remained behind bars Monday.
Lowe was held in lieu of $50,500 bond on charges of felony evidence tampering, carrying a concealed weapon and drug paraphernalia possession.
Bond was set at $50,250 for Cutlip, who was charged with felony drug tampering and drug paraphernalia possession.
Walters was held in lieu of $26,500 on charges of aggravated drug possession and possession of drug abuse instruments.
Tapia was charged with aggravated drug possession, aggravated drug trafficking, receiving stolen property, cocaine possession, possession of drug abuse instruments, possession of a controlled substance and possession of drug paraphernalia.
His bond was set at $100,750.
Reach Lori at 330-580-8309 or firstname.lastname@example.com.
On Twitter: @lsteineckREP


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Summary:The Michigan v. EPA decision is significant for the development of environmental law.
In Michigan v. EPA, the Supreme Court's major environmental decision of 2015, the court struck down an Environmental Protection Agency regulation intended to control emissions of mercury and other hazardous air pollutants from coal- and oil-fired power plants. The court ruled that the agency erred by failing to consider industry's costs of compliance in deciding, at the outset of the regulatory process, whether to develop the regulation. In the short term, the decision will likely cause only a small hiccup in the EPA's progress toward regulating these emissions. Over the longer term, however, the case is a significant recognition of the importance of costs in environmental standard-setting, and illustrates the growing skepticism of some justices about whether it is appropriate—or even constitutional—for the judiciary to defer to agencies' interpretations of federal statutes.
The litigation arose from a set of mind-numbingly complicated provisions of the Clean Air Act (CAA). In 1990, Congress adopted amendments to the CAA creating a new program designed to control air pollution from power plants. Hedging its bets, Congress included language in the amendments directing the EPA to study how effective this new initiative was in controlling so-called "hazardous" air pollutants from power plants. If the EPA determined based on the study that it was "necessary and appropriate" to do more to control hazardous pollutants from power plants, the agency was charged with developing further regulations under the auspices of the EPA's separate hazardous air pollution program.
The EPA completed the study called for by Congress and determined that further regulatory restrictions on power plants were both "appropriate and necessary." It said they were appropriate because mercury and other hazardous pollutants pose a serious risk to human health and the environment, and technology is available to control these emissions; they were necessary because the primary air pollution program targeting power plants had not solved the toxic pollution problem. The EPA did not explicitly consider industry's costs of compliance at this stage of the administrative process. Thereafter, following a set of fits and starts (including two changes in presidential administrations and much litigation), the Obama administration's EPA issued a final regulation for hazardous emissions from power plants. The EPA's regulatory impact analysis concluded that the final regulation would force power plants to bear costs of $9.6 billion per year but produce quantifiable direct and indirect benefits of $37 to $90 billion per year.
The issue before the Supreme Court was whether the EPA erred in failing to consider the costs of compliance at the outset of the regulatory process in deciding whether it was "necessary and appropriate" to develop the regulations. On that issue, in a decision issued June 29, 2015, the court split 5 to 4. Justice Antonin Scalia wrote the opinion for the majority joined by Chief Justice John Roberts and Justices Anthony Kennedy, Clarence Thomas, and Samuel Alito. While recognizing that the statute was ambiguous, Justice Scalia said it was "unreasonable" for the EPA to read the word "appropriate" as not requiring the agency to consider costs, among other relevant factors, in deciding whether to regulate. Accordingly, the court struck down the regulation and sent it back to the agency for further work.
Justice Elena Kagan, joined by Justices Ruth Bader Ginsburg, Stephen Breyer, and Sonya Sotomayor, dissented. Justice Kagan conceded that it was sensible for the EPA to consider costs at some stage in the course of developing the regulation. But she argued that it was "appropriate" for EPA to consider only health effects and technological issues in deciding the initial question of whether to develop a regulation, and to defer consideration of costs to the later stage when the EPA actually formulated the regulation. Without a specific proposed regulation to analyze, she argued, it was impossible to conduct meaningful assessment of potential costs.
The debate between the Supreme Court majority and the dissenters ultimately came down to a dispute over how much to defer to the EPA's reading of the Clean Air Act in light of the court's landmark 1984 decision in Chevron U.S.A. Inc. v. Natural Resources Defense Council, which requires courts to defer to an agency's reasonable interpretation of an ambiguous statute. Justice Scalia said the agency "strayed far beyond" the bounds of a "reasonable interpretation" of the statute. By contrast, Justice Kagan argued that the EPA acted "well within its authority in declining to consider costs" at the initial stage of deciding whether to regulate.
The decision is significant for the development of environmental law for at least two reasons. First, the decision elevates the importance of costs in environmental standard setting, perhaps to the point of creating a virtual presumption that costs must be considered. For decades environmental lawyers and policymakers have debated whether compliance costs should be considered in setting rules to protect public health and the environment. In Michigan v. EPA every justice accepted the idea that costs should be taken into account at some stage of the process of developing this Clean Air Act rule.
Second, the decision appears to signal potential erosion of the Chevron doctrine. Echoing other recent expressions of skepticism from some justices about Chevron's deference mandate, Justice Scalia, while nominally following the command of Chevron, appears to have applied the precedent narrowly. More pointedly, Justice Thomas, in a concurring opinion, raised the question of whether the Chevron doctrine violates the constitutional separation of powers principle by forcing judges to abandon their responsibility to "say what the law is." Because so much environmental law involves questions of statutory interpretation, the weakening or potential overthrow of Chevron could have profound implications for environmental law. It is hard to say whether this move would help or harm the cause of environmental protection given that in Chevron itself the court's stance of deference led it to reject an environmental group's challenge to an EPA decision not to adopt stricter Clean Air Act rules.
In the meantime, the Michigan v. EPA decision likely will not cause much if any delay in the implementation of the new hazardous pollution rules for power plants. The EPA has already calculated the costs and benefits of controlling these pollutants, and to comply with the court's mandate the agency simply needs to go back and incorporate these calculations into a revised assessment of whether it is "appropriate" to regulate. The only open question is whether the new regulation—which many power plant operators are now working hard to comply with—can remain in effect while the EPA revises its analysis, an issue currently pending before the U.S. Court of Appeals for the District of Columbia Circuit.


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Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, including Borrelia burgdorferi, the causative agent of Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing σ70. Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defective-motility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.
Borrelia burgdorferi is the causative agent of Lyme disease and belongs to a group of bacteria called spirochetes. B. burgdorferi cells have a characteristic flat-wave morphology and unique means of motility (9, 10, 19, 35). As a result of its unique morphology and motility, B. burgdorferi is able to traverse viscous gel-like media in which most other flagellated bacteria slow down or stop (30). Consequently, B. burgdorferi may efficiently bore through host tissues, leading to pathogenesis in the joints, nervous system, and heart (19, 30, 54). The motility of B. burgdorferi results from the coordinated rotation of the periplasmic flagella residing between the outer membrane and the cell cylinder (9, 10, 12, 20, 33, 35). The current swimming model suggests that a run occurs when the anterior periplasmic flagella rotate in one direction (e.g., counterclockwise [CCW]) and the posterior flagella rotate in the opposite direction (clockwise [CW]). Reversals occur when periplasmic flagella at both poles of the cell change their direction of rotation. During a nontranslational mode (flex), the periplasmic flagella at both cell poles rotate in the same direction (9, 20, 35).
Bacterial flagella are composed of three major parts: the motor, hook, and filament. The motor can be divided into two functional units, the rotor and the stator. The stator is the torque generator consisting of the MotA-MotB complex. The rotor is composed, minimally, of the MS ring (FliF), the rod, and the switch complex (FliG, FliM, and FliN). In B. burgdorferi, the periplasm-localized flagellar filament consists of the core protein FlaB and the sheath protein FlaA (17, 46). There are 7 to 11 filaments connected to flagellar motors near each cell pole that extend toward the cell body in an organized ribbonlike fashion (9, 10). FlaB is necessary for cell motility and contributes to the distinctive flat-wave cell morphology (12, 38, 43, 51). A mutation in flaB, the switch protein gene fliG2, or the hook protein gene flgE resulted in cells that lack the periplasmic flagella, are nonmotile, and are rod shaped (38, 43, 51). Genome sequence analyses indicate that B. burgdorferi contains homologues of the motility and chemotaxis proteins found in other bacteria (9, 14, 20, 35, 38, 45). However, B. burgdorferi motility and chemotaxis genes are not regulated by hierarchical gene regulatory cascades found in other species of bacteria, e.g., Escherichia coli (1, 9, 18, 51). Genome sequence analysis also indicates that homologues of σ28 and its anti-sigma factor FlgM are not present in B. burgdorferi and that all motility and chemotaxis genes identified to date appear to be transcribed by RNA polymerase containing σ70 (9, 20, 51).
Of at least 25 genes needed to build a functional bacterial flagellum, fliL is one of the genes that is not well understood. The deficiency in information regarding FliL is due in part to the poor homology among bacterial species and a lack of consistent mutational phenotypes. fliL mutants in E. coli or Salmonella enterica have been reported to have a minor defect in swimming motility, but swarming motility was abolished (3). In Caulobacter crescentus, fliL mutant cells are paralyzed, and FliL is required for the cell cycle-dependent ejection of the polar flagellum during the swimmer-to-stalk-cell transition (26). In Proteus mirabilis, fliL mutant cells failed to synthesize flagellin, are nonmotile, and are longer than wild-type cells (5). Recent data indicated that FliL of Rhodobacter sphaeroides is essential for swimming motility (55). Despite these different motility phenotypes, FliL is consistently reported to be associated with the cytoplasmic membrane in each of these organisms (3, 5, 26, 55). For E. coli, the FliL C terminus was also reported to be localized in the periplasmic flagellar stator (3). The B. burgdorferi fliL gene is located between motA-motB and fliM-fliN of the large flgB motility operon (11, 14, 16). It shares poor homology (<10% amino acid sequence identity) with FliL proteins of other bacterial species (34). To date, the function and localization of FliL have not been reported for any spirochetes. Detailed structural and genetic analyses of FliL are required to better understand flagellar assembly and the mechanism of bacterial motility at the molecular level.
Recently, cryo-electron tomography (cryo-ET) was utilized to determine the molecular architecture of the spirochete flagellar motor in situ (24, 25, 40, 41). These studies suggested that the overall spirochetal flagellar motor structures (the MS ring, the C ring, and the export apparatus) are similar to those of other bacteria, e.g., E. coli (9, 25, 31, 33, 40, 41, 47). B. burgdorferi is representative of highly motile and invasive bacterial pathogens and has a small cell diameter (∼0.3 μm) and an orderly arrangement of multiple flagellar motors, making it ideal for the structural analysis of the intact flagellar motor in situ (10, 33, 41). However, B. burgdorferi remains challenging to manipulate genetically despite significant progress that has been made in developing both allelic exchange and transposon mutagenesis. Specifically, the currently available antibiotic resistance cassettes utilized for B. burgdorferi gene inactivation or the transposon mutations can cause polar effects on downstream gene expression, complicating interpretations of results obtained from such mutants (23, 35, 41).
In this communication, we describe the development of a novel procedure to generate nonpolar mutations in B. burgdorferi genes. Using this methodology, we characterize a flagellar protein, FliL. Our studies indicate that B. burgdorferi nonpolar fliL mutant cells exhibit defective motility. Surprisingly, we discover that the periplasmic flagella of fliL mutant cells are frequently tilted toward the cell pole instead of the normal orientation toward the cell body found in wild-type cells. The fliL mutant has a loss of density in a region between the proposed rotor and stator, as determined by cryo-ET (41). Wild-type motility, the periplasmic flagellar orientation, and motor structural defects are restored when the mutant is complemented in cis. Based on the observed phenotypic changes and the apparent location of FliL within the flagellar motor, the possible relationships of FliL with other flagellar proteins and the resulting effects on flagellar orientation are discussed.
Bacterial strains and growth conditions.High-passage, avirulent B. burgdorferi sensu stricto strain B31-A and flaB mutant strain MC-1 were described previously (6, 43). Cells were grown in Barbour-Stoenner-Kelly II (BSK-II) medium at 35°C in a 2.5% CO2 humidified incubator as described previously (44, 52). BSK-II medium for plating contained 0.6% agarose.
Construction and complementation of the fliL mutant.The fliL gene is separated from motB and fliM by 48 and 37 bp, respectively (14). The 48-bp intergenic sequence between motB and fliL possess a ribosomal binding site. The fliL gene (gene locus BB0279, a 537-bp gene) was inactivated by replacing fliL with the aadA coding sequence for streptomycin-spectinomycin resistance (13) by using overlapping PCR, as schematically shown in Fig. 1. PCR was used to amplify 3 regions of DNA in 3 steps. In the first step, each DNA region was amplified separately by using PCR pairs P1-P2 (5′-flanking DNA, motB), P3-P4 (aadA coding sequence), and P5-P6 (3′-flanking DNA, fliM). Primers P2, P3, P4, and P5 (Table 1) contain several overlapping base pairs, as indicated by different colors in Fig. 1. During step 2, a PCR product was obtained by using primers P1-P4 and the purified DNA products for motB and aadA as templates. In step 3, the final PCR product was obtained by using primers P1-P6 and the purified DNA products of motB-aadA and fliM as a template to amplify the motB-aadA-fliM DNA construct. The final PCR product yielded a 2,725-bp product that was gel purified and cloned into the pGEM-T Easy vector (Promega Inc.). The integrity of the fliL inactivation plasmid was confirmed by PCR and restriction mapping. PCR-amplified DNA was electroporated into B31-A competent cells and plated onto BSK-II solid medium containing 80 μg/ml streptomycin, as described previously (44). Resistant clones were analyzed by PCR and Western blotting using specific antisera raised against recombinant FliL (see below).
Complementation of the ΔfliL mutation.The B. burgdorferi flgB promoter (PflgB) and the fliL gene were PCR amplified separately by using primers P7-P8 and P9-P10, respectively (primer sequences are presented in Table 1). Overlapping PCR was used to construct PflgB-fliL in such a way that the promoter drives the expression of fliL. This construct also contained engineered 5′-SpeI–3′-NotI ends. The PflgB-fliL DNA was then ligated into the SpeI-NotI sites of a suicide vector, pXLF14301, that contains a gentamicin resistance cassette (PflgB-aacC1) (39, 58). The resulting construct was used to complement the fliL mutant (ΔfliL) by inserting it into a heterologous chromosomal site between the BB0445 and BB0446 genes as described previously (39, 58). Plasmid DNA was electroporated in ΔfliL cells and plated, and the transformants were selected with 40 μg/ml gentamicin plus 80 μg/ml streptomycin. Resistant clones were analyzed by PCR and Western blotting for the integration of PflgB-fliL within the BB0445 and BB0446 genes and restoration of FliL synthesis, respectively.
Protein preparation and antibody production.To express recombinant FliL (178-amino-acid protein), we PCR amplified the coding sequence without the predicted transmembrane domain encoded by the first 150 bp. The PCR primers (5′ to 3′) were T-R-FliL-F (5′-GTGTCTAAAATGGTGGTAAGCC-3′) and T-R-FliL-R (5′-TTACATATCAAAAATATCAATTTGGG-3′). The amplified 386-bp DNA fragment was ligated into an E. coli expression vector, pTrc-His TOPO, using TA cloning (Invitrogen Inc.). The resulting vector carrying fliL was transformed into an E. coli host strain, DH5α, and induced by using 0.25 mM isopropyl β-d-thiogalactoside for 4 h at 37°C. The recombinant protein was purified by using Ni2+ affinity chromatography according to the manufacturer's protocol (Invitrogen Inc.) and dialyzed against phosphate-buffered saline. Rabbits were immunized with approximately 400 μg of dialyzed protein to raise specific antiserum according standard methods (Alpha Diagnostic International).
Gel electrophoresis and Western blot analysis.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with an enhanced chemiluminescence detection method (GE Healthcare) were carried out according to the manufacturer's instructions. Growing B. burgdorferi cells were pelleted, washed twice with phosphate-buffered saline, and resuspended in the same buffer. The protein concentration of cell lysates was determined by a Bradford protein assay kit (Bio-Rad Inc.). Unless otherwise noted, 10 μg of lysates was loaded into each lane of an SDS-PAGE gel and subjected to Western blotting using specific antibodies. Monoclonal and polyclonal antibodies kindly provided by other investigators included the following: monoclonal anti-B. burgdorferi FlaB (H9724) from A. G. Barbour (University of California, Irvine, CA), polyclonal anti-E. coli FliM from D. Blair (University of Utah, Salt Lake City, UT), and polyclonal anti-B. burgdorferi MotB from J. Carroll (RML, NIH, Hamilton, MT). The reactivities of those antibodies to B. burgdorferi FlaB, FliM, and MotB were reported previously (8, 35, 43, 46, 51).
Swarm plate motility assays.Swarm plate motility assays were performed as described previously (43, 44). Approximately 1 × 106 cells in a 5-μl volume were spotted onto a 0.35% or 0.5% agarose plate containing plating BSK medium diluted 1:10 in Dulbecco's phosphate-buffered saline. Since B. burgdorferi is a slow-growing organism (8- to 12-h generation time), plates were incubated for 5 days at 35°C in a 2.5% CO2 humidified incubator. The paired Student t test was used to compare wild-type and fliL mutant cell swarm diameters.
Microscopy and computer-assisted motion analysis.Live B. burgdorferi cells were observed under a dark-field microscope (Zeiss Axio Imager M1) connected to an AxioCam digital camera. Exponentially growing cells were mixed with 0.5% 400-mesh methylcellulose (Sigma-Aldrich Co.) and video recorded at room temperature (23°C). Cell swimming behavior and velocity were determined by using Axivision software. Results are expressed as velocity (mean distance in μm traveled by a given strain per second). At least eight cells from each strain were analyzed. The paired Student t test was used to compare wild-type and fliL mutant cell swimming velocities.
Cryo-electron tomography of frozen-hydrated fliL organisms.Cryo-ET was carried out as described previously (41). Briefly, the B. burgdorferi culture was deposited onto a glow-discharged holey carbon electron microscopy (EM) grid, blotted, and rapidly frozen in liquid ethane. The frozen-hydrated specimens were imaged at −170°C by using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a 4,096-by-4,096-pixel charge-coupled-device (CCD) camera (Tvips GmbH, Germany). The microscope was operated at 300 kV with a magnification of ×31,000. Low-dose single-axis tilt series were collected from each bacterium at a 5-μm defocus with a cumulative dose of ∼100 e−/Å2 distributed over 65 images with an angular increment of 2°, covering a range from −64° to +64°. The tilt series images were aligned and reconstructed by using the IMOD software package (32). In total, 98, 123, and 149 cryo-tomograms from the wild type, an fliL mutant (ΔfliL), and a complemented fliL+ strain were reconstructed, respectively. The effective pixel size was 5.6 Å.
3-D subvolume averaging of the flagellar motor.The subvolume processing of flagellar motors was carried out as described previously (41). The positions and orientations of each periplasmic flagellar motor in each tomogram were determined manually. In total, 711 flagellar motor subvolumes (256 by 256 by 256 voxels) containing the flagellar motors were extracted from 123 tomograms of fliL mutant cells and were further aligned to generate the three-dimensional (3-D) structure. In addition, 743 flagellar motors from complemented fliL+ cells were selected for 3-D analysis. Finally, 591 flagellar motors from wild-type cells were selected as a control for the 3-D analysis.
3-D visualization of the cell and flagellar motor.Tomographic reconstructions were visualized by using IMOD (32). One organism of the B. burgdorferi fliL mutant strain was segmented manually by using Amira 3-D modeling software (Visage Imaging). The 3-D segmentations of flagellar filaments, peptidoglycan, outer surface proteins, and outer and cytoplasmic membranes were manually constructed. The surface model from the averaged flagellar motor was computationally inserted into the segmented model of the organism.
Development of a novel methodology to inactivate a gene without causing polar effects.A common problem of insertion mutagenesis using antibiotic resistance cassettes is a polar effect on genes downstream from the targeted gene. Because of the polar effect, interpretations of results obtained from such a mutant are complicated (23, 35, 41). Investigators commonly use a B. burgdorferi flgB or flaB promoter fused to the kanamycin (PflgB-Kan), streptomycin (PflgB-aadA), or gentamicin (PflgB-aacC1) resistance cassette to inactivate a gene (50). Recently, we reported the development and use of promoterless cassettes (PL-Kan or PL-aadA) with the hypothesis that the use of a cassette without a promoter decreases the chance of having a polar effect (56). Here we report the further development of a novel methodology to inactivate B. burgdorferi genes. PL-Kan or PL-aadA contained no promoter but had an intact ribosomal binding site and a transcription start site of the flgB promoter PflgB (6, 18, 56). Although those cassettes do not cause a polar effect, we further refined the PL-aadA cassette. We hypothesized that if a B. burgdorferi gene next to or away from a promoter is expressed, the replacement of the gene with an antibiotic resistance gene coding sequence should also be expressed and confer resistance to that antibiotic. Accordingly, we replaced the fliL gene with the antibiotic resistance gene (only the aadA coding sequence from the ATG start to the TTG stop codon) in a way that aadA uses the targeted gene's RNA polymerase machinery to be expressed by maintaining transcriptional readthrough. Streptomycin-resistant clones were confirmed by PCR for the integration of the aadA coding sequence replacing fliL, as expected (not shown). Polyclonal antiserum was raised against purified recombinant 6×His-FliL, and the inactivation of the fliL gene was confirmed by Western blotting. As shown in Fig. 2, the antibody failed to detect a 20-kDa protein band in fliL mutant (ΔfliL) cells, indicating the inactivation of fliL, whereas this protein was detected in wild-type and complemented cells (see below). These results confirmed our hypothesis that an antibiotic resistance gene can be expressed by using the targeted gene operon's transcription machinery and confer resistance once it undergoes homologous recombination.
Determination of polar effect and complementation of ΔfliL cells.We performed Western blotting to determine if the insertion of the "strep" (aadA coding sequence) marker resulted in an altered expression of genes that are immediately upstream or downstream of fliL. The fliL gene is located in the middle of the large flgB motility operon containing 26 genes (9, 14, 16, 18, 20). The motB and fliM genes are immediately upstream and downstream, respectively, of fliL (Fig. 1). Western blotting with a polyclonal anti-FliM or anti-MotB antibody indicated that FliM or MotB protein synthesis was not altered in the ΔfliL mutant cells (Fig. 2). These results indicated that the replacement of the fliL gene with aadA did not cause a polar effect. Thus, this strep marker can be used to inactivate genes that are within an operon without introducing a polar effect.
Although the fliL mutant cells did not exhibit a polar effect, we complemented the mutant in cis by integration in a heterologous chromosomal location to prevent overexpression (39, 58). Furthermore, bacterial flagellar proteins maintain a stoichiometric relationship with other flagellar proteins (27, 42), and maintaining their ratio is critical for restoring a mutated gene's function (15, 57; our unpublished observations). To complement the fliL mutant, we fused fliL with the flgB promoter (PflgB) and then inserted the PflgB-fliL construct within the BB0445 and BB0446 genes using a suicide vector that was reported previously to complement B. burgdorferi mutants in cis (39, 58). The PflgB-fliL insertion was in a region where both the BB0445 and BB0446 genes converge; thus, the inserted DNA was downstream of both genes and less likely to affect their expression (39, 58). The genomic integration of PflgB-fliL within BB0445-BB0446 was confirmed by PCR (not shown). The restoration of FliL protein synthesis was determined by Western blotting. FliL protein synthesis in the complemented fliL (fliL+) cells was restored and at the same level as that of wild-type cells, indicating that FliL was not overexpressed (Fig. 2).
The fliL mutant is defective in motility.B. burgdorferi FliL is predicted to contain a transmembrane domain at the N-terminal 33 to 51 amino acid residues using the secondary-structure topology prediction program TMpred (22; not shown). The gene function for fliL has not been reported for B. burgdorferi or any other spirochetes. We performed motility assays using 0.35% and 0.5% agarose plates in order to determine the effect of FliL on cell motility and compared the swarm diameters of wild-type, ΔfliL, and complemented fliL+ cells. A nonmotile periplasmic flagellinless flaB mutant was used as a control (43). Motile cells swarm out from the initial site of inoculation as they metabolize nutrients. In many species of bacteria, swimming and swarming are assayed on plates containing low-concentration (0.3%) and high-concentration (0.5 to 0.7%) agar media, respectively (3, 21). However, in the spirochetes/B. burgdorferi, swarming is assayed on plates containing 0.35% agarose (35–38, 43, 45, 51). Thus, to be consistent, we continue to use the term "swarming" when B. burgdorferi cells were assayed on plates containing 0.35% agarose. Unlike other species of bacteria (e.g., E. coli and S. enterica), B. burgdorferi wild-type cells do not swarm on 0.5% agarose (Fig. 3a). However, when cells were inoculated in 0.35% agarose, the fliL mutant cells exhibited significantly defective swarming motility compared to that of the wild-type parental cells (P value of 0.002). As shown in Fig. 3b and c, the fliL mutant cell swarm diameters were similar to those of the flagellinless nonmotile flaB cells. The motility defect exhibited by the fliL mutant cells was restored upon complementation (Fig. 3b and c). These results indicate that FliL is required for full motility and that the phenotype was attributed solely to the fliL mutation, and there was no secondary alteration.
Furthermore, when the fliL mutant cells were grown in BSK-II broth, we noticed that these cells sometimes twisted with each other and formed small clumps at the bottom of the culture tubes, which is indicative of defective swimming motility. Dark-field microscopy coupled with video analysis of swimming cells demonstrated that wild-type B. burgdorferi cells ran, paused (flexed), and reversed. The fliL mutant cells also ran, paused, and reversed; however, their flexing was brief and did not completely distort the cell morphology or bending at the center of the cell, as seen for the wild-type cells (19, 35, 45). B. burgdorferi cells do not efficiently swim in liquid growth medium, but the swimming velocity increases in a gel-like medium, such as one containing methylcellulose (4, 7, 19, 30, 38, 44). Accordingly, wild-type and mutant cells were mixed with 0.5% methylcellulose, and cell swimming velocities were determined. The ΔfliL mutant cells had a swimming velocity of 3.4 ± 0.49 μm/s, whereas the wild-type cells swam at a significantly higher rate, with a velocity of 5.5 ± 0.19 μm/s (P = 0.01). The complemented cells restored the swimming motility (velocity of 4.8 ± 1.2 μm/s). Taken together, these results indicate that the ΔfliL mutant cells are defective in motility. In vitro growth curve studies confirmed that ΔfliL mutant cells had no appreciable growth defect relative to wild-type or complemented fliL+ cells, eliminating the possibility that phenotypic differences were a consequence of in vitro growth differences between the clones (not shown).
Abnormal orientation of periplasmic flagella in the fliL mutant.The morphology of the B. burgdorferi fliL mutant was examined by dark-field microscopy and was found to be indistinguishable from that of wild-type cells (not shown; see below). Furthermore, FlaB expression levels, which partly determine the B. burgdorferi wavelike cell morphology (12, 20, 38, 43, 49, 51, 53), were similar between the wild type and the fliL mutant, as confirmed by Western blotting (Fig. 2). Cryo-ET of intact organisms indicated that the overall cellular morphology and the flagellar motor structure of ΔfliL cells are similar to those of wild-type cells (Fig. 4). The deletion of fliL did not alter the formation or assembly of periplasmic flagella. However, we found that the ΔfliL cells showed an unusual structural phenotype: of five periplasmic flagella visible in the 3-D reconstructions of the cell shown in Fig. 4, three of them were directed toward the cell pole (see arrows in Fig. 4d and f and see Movie S1 in the supplemental material) instead of being oriented toward the middle of the cell body, as found for the wild-type parental cells (Fig. 4c and e). We examined more than 40 cells from each strain to determine the normal and abnormal/irregular periplasmic flagella in each cell (Table 2). On average, 26.4% of the periplasmic flagella in each ΔfliL mutant cell were oriented toward the cell pole (irregular/abnormal orientation) (Table 2), whereas only 1.7% of periplasmic flagella from each wild-type cell were oriented toward the cell pole (Table 2). Similar to the wild-type, the complemented fliL+ cells restored the phenotype, as only 3.6% of each cell's periplasmic flagella were detected to exhibit the abnormal orientation (Table 2). However, although the periplasmic flagellar motors of the ΔfliL cells are located in the cell poles, as seen for the wild-type cells, the abnormally oriented flagellar filaments are shorter (0.3 to 2 μm), ending at the cell pole, than normal flagella (∼6 μm), extending toward the cell body (Fig. 4d).
FliL localization in the flagellar motor.The molecular architecture of the periplasmic flagellar motor in the ΔfliL mutant was determined by tomographic subvolume alignment and averaging. The 711 motors extracted from 123 B. burgdorferi cells were used to generate a 3-D flagellar motor structure from the fliL mutant at a resolution of ∼4 nm. Without imposing any rotational symmetry, the structure preserves key asymmetric features, as described previously for the structure of wild-type cells (Fig. 5a), including the visualization of the hook and the curvature of the stator embedded in the cytoplasmic membrane (41). The overall structure of periplasmic flagellar motors from ΔfliL cells closely resembles that of wild-type cells (Fig. 5a and d), as shown in their central sections, which illustrate the locations of the putative hook, rod, MS ring, stator, C ring (FliG, FliM, and FliN), and export apparatus.
Despite the overall similarity between the motor structures of ΔfliL and wild-type cells, there are clear distinctions between them (Fig. 5a and b, orange arrows). The periplasmic flagellar motors of the ΔfliL cells lacked a density just outside the collar region of the rotor (Fig. 5b and e), which is clearly visible in wild-type cells (see orange arrows in Fig. 5a) or complemented fliL+ cells (Fig. 5c). This feature appears to extend from the cytoplasmic membrane into the periplasmic space and is located between the proposed bowl-shaped rotor and the surrounding stator structures (Fig. 5d) (41). Therefore, the extra density in flagellar motors of wild-type organisms corresponds to the FliL protein (labeled L in Fig. 5d and f).
The fliL gene has been characterized in E. coli, S. enterica, Caulobacter crescentus, Proteus mirabilis, and, recently, Rhodobacter sphaeroides (3, 5, 26, 55). FliL of R. sphaeroides was reported previously to be essential for flagellar rotation and modulates the motor function through an interaction with MotB (55). An fliL mutant of E. coli or S. enterica was found previously to be defective in swarming motility (on 0.5 to 0.7% agar plates) but had a minor defect in swimming motility (in 0.3% agar) (3). Based on their findings, Attmannspacher et al. proposed previously that in E. coli and S. enterica, "flagellar motors experience a higher torque during swarming motility owing to an increased proton motive force, and that FliL allows the flagellar rod to withstand the increased torsional stress" (3). Because of this stress, the mutant cells had a hook-filament-ejection phenotype (3). For C. crescentus, an fliL mutant was found to be paralyzed, and the FliL protein was reported to be required for the cell cycle-dependent ejection of the polar flagellum during the swimmer-to-stalk-cell transition (2, 26, 28, 48). A P. mirabilis fliL mutant was reported to be nonmotile, failed to synthesize flagella, and had an elongated-cell phenotype (5). Furthermore, based on its predicted location and phenotype, Belas and Suvanasuthi suggested that FliL may be responsible for the transmission of signals from the exterior flagellar filament into the cell (5). Clearly, FliL has diverse functions in these organisms. However, there is a consensus that FliL is a transmembrane protein associated with the flagellar motor (3, 26, 29, 55). Based on the findings with the P. mirabilis and E. coli/S. enterica fliL genes, two models were proposed for the location of FliL (3, 5). In both models, FliL is predicted to be a part of the flagellar motor.
B. burgdorferi ΔfliL cells exhibited a significant defect in swimming (in methylcellulose) and swarming (on 0.35% agarose plates) motility despite the fact that the amounts of the flagellar filament protein FlaB and the motor protein MotB were not altered in the ΔfliL strain. The defective-motility phenotypes are similar to those found previously for swarming fliL mutants of E. coli/S. enterica (3). Whereas different mechanisms were proposed for defective motilities in different organisms (3, 5, 26, 55), we propose that periplasmic flagella with abnormal orientations oppose smooth swimming mediated by the standardly oriented flagella, resulting in defective motility in B. burgdorferi. The wavelike morphology of B. burgdorferi, which is dictated at least in part by the presence of flagellar filaments, was not altered in ΔfliL cells (Fig. 2) (9, 12, 20, 38, 43, 51, 53). Cryo-ET of B. burgdorferi ΔfliL cells indicated that the periplasmic flagella are connected to the hook-basal body (Fig. 4). However, we were surprised that 26.4% (versus only 1.7% in the wild type) (Table 2) of the motors/periplasmic flagella were oriented toward the cell pole rather than extending toward the middle of the cell body. This remarkable phenotype has not been observed for previously characterized spirochetal flagellar mutants (24, 41). FliL is therefore likely to play a role in defining the orientation of periplasmic flagella in B. burgdorferi (Fig. 4), although a mechanism is currently unknown. It is also unknown why 26.4% but not all periplasmic flagella of each ΔfliL cell were tilted toward the cell pole. We speculate that FliL and its interacting protein(s) may be responsible for the orientation of periplasmic flagella. Nevertheless, the altered flagellar orientation is likely to be the underlying reason for the defective-motility phenotype observed for the fliL mutant cells (Fig. 3). In contrast, the lack of FliL tends to fracture the flagellar rod in swarmer cells of S. enterica (3). Furthermore, the defective motility exhibited by R. sphaeroides fliL was proposed previously to be mediated through modulating the MotB function (55). Therefore, our data reinforce the notion that the mechanisms of altered motility observed for B. burgdorferi and these other bacteria are likely to be different, even though fliL mutants of B. burgdorferi and S. enterica share similar defective-motility phenotypes.
Multiple copies of FliL are located between the rotor and stator at the cytoplasmic membrane in B. burgdorferi (Fig. 5), although there is a lack of detailed information about its shape or stoichiometry. The deletion of fliL has little effect on flagellar assembly and the overall molecular architecture of the flagellar motor. However, there is clear evidence of an abnormal periplasmic flagellar orientation and defective motility of fliL mutants. Recently, Kudryashev et al. speculated that a protein which links the rotor with the stator is likely to be FliL (32). Our data provide the first structural evidence that B. burgdorferi FliL is located between the rotor and stator which in some manner affects the periplasmic flagellar orientation and, thus, swimming efficacy.
We thank Nyles Charon, Steven Norris, Rasika Harshey, Everett Pesci, and Melanie Sal for comments on the manuscript. We thank D. Blair, A. Barbour, J. Carroll, S. Samuels, U. Pal, X. Li, and E. Fikrig for reagents.
This research was supported by an East Carolina University Research and Development start-up fund to M.A.M. and grants R01AI087946 from the National Institute of Allergy and Infectious Diseases (NIAID) and AU-1714 from the Welch Foundation to J.L.
- Received 11 February 2011.
- Accepted 14 March 2011.
- Accepted manuscript posted online 25 March 2011.
† Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00202-11.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.


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Intro Wp Rocket Coupon Codes
Don't you Just despise slow sites? Well, you are definitely not alone. Your visitors hate them too– and, to make things worse, so does Google. That is right, if you want your site to rank highly in search engines, sell more goods, generate more leads, earn more revenue, or just deliver a more positive user experience, it must load quickly. Wp Rocket Coupon Codes
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So how Can you make your WordPress site quicker? Well, if you are still using a generic shared hosting company, one of the best options will, naturally, be to simply switch to a greater performance hosting company. But what if you just have not got the time or money to make the change?
Well, Luckily, there's something else you can do that is extremely quick and simple — something which needs next-to-no technical know-how whatsoever! I am talking, of course, about using a caching plugin.
Today, I will be taking an comprehensive look at a few of the primary caching plugins round. WP Rocket. In the next review, I'll show you by exactly how much — and how easily — it can speed up your website (using real-life evaluations and actual data).
What is WordPress Caching?
Caching Involves generating — and saving — a readymade static (i.e. unchanging) variant of your site in your own server ready to display to people at a moment's notice. This reduces the need for your website (or the user's browser) to communicate back and forth with the server to assemble the behind-the-scenes information that creates your site's web pages.
With less Communication happening between servers, and less data being loaded whenever your website is accessed, the time it takes to load your articles and pages is considerably reduced.
WP Rocket Will possibly.
· Cache all of the posts and pages on your website
· Enable browser caching
· Defer loading of JavaScript documents
· Decrease bandwidth usage with gzip compression
· optimise Google fonts
· minify and concatenate CSS and JS files
· Defer loading of pictures.
You might Be wondering what happens in the event that you publish a new blog article on a cached website, because, paradoxically, an unchanging cached version of your site wouldn't take this recently created data into consideration.
Not so! WP Rocket will regenerate the cached pieces of your website each time you update your content, thereby ensuring your visitors will always see the most up-to-date variant of your website.
Features of WP Rocket
It's worth Pointing out that, when it comes to features alone, WP Rocket compares pretty Favorably using its competitors. Granted, you're probably more interested in just how much faster your website will be after installing WP Rocket (and rightfully so), however it is nice to know you're not missing out on any important features.
With WP Rocket, you receive page caching, cache preloading, gzip compression, browser caching, Google Fonts optimization, picture and video Lazy Load, minification, plus a whole lot more.
If you do Choose to explore the additional settings and choices WP Rocket supplies, you'll see it has a couple of tricks up its sleeve to get potentially improving speed much further, such as the choice to only load media files (such as pictures ) whenever the visitor reaches them on a page (a feature known as Lazy Loading).
One of The highlights of WP Rocket is its easy installation procedure, making it a pretty much fool proof caching choice for busy WordPress webmasters. It's called being'childishly simple to set up' — something users who just want to get the job done and move on will almost certainly appreciate. Wp Rocket Coupon Codes
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Actually, Once the plugin was uploaded and activated on your site, the cache will be automatically generated whenever someone visits the site. Everybody visiting your site thereafter should immediately begin experiencing significantly quicker loading times. Simple!
WP Rocket Aims to remove all such confusion by being automatically up-and-running right from the box without being overly rigid that it is not able to deliver great results. After tripping WP Rocket, you'll see just a couple of tabs on the admin panel, each with just a couple of easy-to-understand settings.
You can See the rest of the basic settings display here. While there are a few more options to work with, the plugin works just fine without any of these enabled. This truly is plug'n' play caching for WordPress!
Of Course, it is suggested that you read the online documentation to actually see what this plugin can do and just how much extra performance you can squeeze out of it, however, for those in a rush (or with a zero technical curiosity from the ins-and-outs of caching), it is safe to say that speeding up your website with WP Rocket couldn't be made easier.
FAST AND Effortless SETUP
The primary Selling point of WP Rocket is how easy it makes allowing caching on your website. Together with many other caching plugins, you are faced with the sometimes daunting task of understanding — and filling out — several pages of settings and options. Based on how you configure them, you could easily end up inadvertently doing something somewhat screwy (and even, in rare cases, inadvertently breaking your site).
WP Rocket Is also compatible with content delivery network (CDN) services. if your site is large enough to warrant distributing its files to multiple servers across the world for faster loading times, you will find WP Rocket more than accommodating.
Custom Made CACHE LIFESPAN
There is In addition, the choice to modify the default cache expiry interval. But, unless you are displaying particularly fast-changing or significant external dynamic content on your site (for instance, critical content from societal networking feeds), the default option 24-hour lifespan should be fine.
Cache — Basic Cache Options
The Cache tab enables you to control How your page caching functions at a fundamental level. you're able to. Wp Rocket Coupon Codes
· Include/exclude mobile users from viewing cached pages.
· Include/exclude logged-in users from seeing cached pages.
· Enable caching on HTTPS pages.
· Decide how long a page stays cached before the cache gets cleared (the default is 10 hours).
Most of the timeyou can Render these as the defaults.
Media — Lazy loading + More
In the Media tab, you can empower Lazy loading for images and/or videos, as well as disable WordPress emojis and embeds.
One feature That's really nice here's the choice to replace YouTube video iframes with a static preview picture. If you embed a lot of YouTube videos on your site, this is a excellent way to speed up your site without sacrificing much, if any, usability.
Document Optimization — Minification Along With Concatenation
The File Optimization tab is Where you are able to set up WP Rocket's minification and concatenation performance.
· Minification — shrinks the size of your website's code by removing unnecessary characters (like whitespace) without changing any functionality.
· Concatenation — reduces the number of files on your site by combining multiple CSS/JS files into a single.
You can also set up some other Settings such as.
· Eliminate gender-blocking CSS/JS (PageSpeed Insights is notorious for yelling at you concerning this problem ).
· Combining Google Fonts files (useful if you use multiple Google Fonts on your site).
· Remove query strings out of static sources (this you will not have a major effect, but it can improve your GTmetrix score, should you really care about that).
I recommend Enabling as a number of these settings as you can. I say as possible because some of the settings for CSS or JS might cause issues with your site (this is not WP Rocket's error — it's almost always a possible issue with minification/concatenation).
What I enjoy is that WP Rocket Makes this clear when you go to activate one of those settings.
Three cheers for good UX Layout!
Preload — Preload Your Own Cache File
Without Preloading, your website only"assembles" the cache for a page when someone actually visits the page. That means the first visitor after the cache lifespan expires does not observe the cached version (because the cache has not been built yet).
Cache Preloading lets you guarantee all visitors see the quick-loading cached page by"warming" yourself, instead of waiting for a person to see the page.
One of those Benefits of all WP Rocket is that it provides you two different ways to preload your cache, whereas a few caching plugins do not even provide one.
· Sitemap preloading — Reload your cache according to all the pages in your sitemap.xml file. It's low impact, but the disadvantage is that it doesn't cache newly published or edited posts.
· Preload bot — mechanically crawls URLs after you publish/edit a post to make sure that recently edited/published content can be preloaded. The only downside is that it's a little bit more resource intensive, which could cause issues on"weak" servers. Wp Rocket Coupon Codes
You can choose to use zero, One, or both approaches to preload your cache, depending on your needs.
Database — Clean Up Your Database
As your website ages, your Database will pick up all types of"gunk" in the form of revisions, trashed opinions, transients, etc..
This area helps you wash out All that unnecessary crap from the database with one click.
Personally, I find this to be The least exciting area of WP Rocket because there are loads of free plugins which do a fantastic job cleaning your database. But I guess the key advantage here is that WP Rocket eliminates the requirement for you to put in another plugin.
CDN — Integrate With Your CDN
If you use a CDN to speed up The worldwide delivery of your site, WP Rocket can help you.
· Rewrite your URLs to use your CDN
· Exclude specific files from your CDN
While you can find free Plugins to do the first component, the second part, plus the fact that you can reserve CNAMEs for particular document types, provides you more flexibility than most of those free options.
For example, you could use one CNAME for your images, and yet another for the remainder of your files.
If you are using Cloudflare, WP Rocket also comprises a dedicated Cloudflare add-on which could help you get set up with Cloudflare and control certain facets of this Cloudflare service right from the WP Rocket interface.
And that pretty much sums up The brand new WP Rocket 3.0 interface!
Through The plugin settings, you can even enable caching for both mobile users and logged-in users — all with a simple click. After moving on to this'advanced' tab, however, things begin to get a little more complicated.
To give you even greater control over how caching Functions on your website, you're also free to exclude pages from the cache with the addition of their URLS to the'never cache the following pages'
Through The CDN tab, you can configure the plugin to work together with your content delivery system of choice, whereas the tools section provides you fast access to the clear and preload cache buttons.
What's More, in the event you get stuck, the FAQ and service tabs make it incredibly easy to access the online documentation, and also open a support ticket from inside your WordPress dashboard — a beautiful touch!
In General, WP Rocket intends to be all things to all people. On one hand, it is very easy to set up and begin using on your site with default settings which actually produce good results, nevertheless, on the flip side, there is also an appreciably large choice of settings and options to appeal to more advanced users with specific needs, or those who need more control over how caching functions on their website.
By Default, each time you print a new article or add fresh content to your site, WP Rocket will automatically regenerate that portion of this cache. Not only that, but will also refresh the related areas of the cache (such as the archive pages, menus, or widgets). Wp Rocket Coupon Codes
Should You need to refresh everything from the cache, though (not just newly updated or altered content), you can force WP Rocket to regenerate it from scratch. Going a step farther, you will also find options to have unrelated parts of your website automatically regenerated when a new post, page, product, comment, or another kind of content gets added.
field. (Note. Shopping cart and checkout pages are automatically excluded from the cache as part of their eCommerce service of WP Rocket.)
Documentation And Support
Even though Using WP Rocket from the box is quite straightforward, should you get stuck or need to know more about the ins and outs of how it works, there is quite a little high tech documentation provided — including a getting started section, different tutorials, helpful FAQs and a handy troubleshooting section.
As long As your license is still active, you can even open tickets with support to receive whatever WP Rocket-related assistance you need.
My WP Rocket Testing Results
As Mentioned at the top of this guide, I had excellent results from using WP Rocket to cache my WordPress site. By testing the rate of my website using GTmetrix — using the default settings of WP Rocket and uploading and activating the plugin prior to generating the cache and conducting the tests — the ordinary page loading times dropped from 2.2 minutes to 0.9.
The Website I tested the plugin on was a WordPress blog with the free Twenty Twelve motif, with about 80 posts and 37 plugins. The site is hosted on an entry level shared hosting program from HostGator, and does not have any CDN in drama.
No two WordPress sites are the same, however, so that these results might not be typical for many sites. They do, however, show that a very fundamental WordPress site on the lowest priced hosting will almost certainly be able to achieve significantly improved load times using the plugin.
Other Methods to Speed Up Your WordPress Website
Whether You opt to use WP Rocket in your site or not, there are a number of different ways to accelerate your WordPress site that is well worth mentioning, such as.
· Deactivating all redundant plugins (many plugins will considerably slow down your website )
· Assessing and optimising all images
· Using a CDN support
· Upgrading to a WordPress-specific high-performance hosting company, such as WP Engine or Flywheel(if your budget allows).
All of These are well worth trying in the event that you haven't already.
WP Rocket Pricing
With Prices starting at only $39 for use on one WordPress site, WP Rocket is an inexpensive option for most. There are also some other strong free options available — such as WP Super Cache, also, for the technically minded, W3 Total Cache (to mention but two of many). If you're searching for something which's exceptionally easy to set up and use, though, and also provides access to a very helpful premium support desk, then WP Rocket is a strong contender.
The complete Pricing choices are as follows. Wp Rocket Coupon Codes
· Personal. $39 (1 sites)
· Company. $99 (3 sites)
· Pro. $199 (unlimited sites)
All Options include one year's access to plugin upgrades and service. Following this period, you may continue using the plugin or renew your permit to keep access. There's also a 30-day refund on offer if you are not entirely pleased with the plugin later purchase.
WP Rocket Is really very striking. Not only is it extremely easy to use, it also provides great results.
Most of The activities you'll be able to execute to optimise the speed of your website take considerable time and effort — whether that is optimising media documents, reviewing and consolidating your plugins, then migrating to a different web host or changing themes. WP Rocket, on the other hand, can be implemented to your website in just a couple clicks.
What's? More, if you're a WordPress freelancer, if helping customers with their present websites or creating websites from scratch, having something like WP Rocket on your toolkit could be of huge benefit. In just a couple of minutes, you will have the ability to accelerate a customer's site and provide them tangible results (in the shape of'before and after' online speeds tests), also, when the very best way to separate yourself from the competition is to provide more value to the client, things like this will really help.
As a Data-backed article over on Moz sets it. "Fast websites have more visitors, who see more pages for longer periods of time, who return more frequently, and are typically more inclined to purchase products or click ads."
Due to Its quick set-up process, speeding up your website with WP Rocket is an easy triumph in the war against slow load times.
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Other people sleep to think about is the bed of the Millennium Falcon. You will require a round mattress, foam to generate the side, fabric and fabric paint. Full bed with Sci-Fi style lights around the floor. You'll find more information online. This bed will definitely require you to anonymous galaxies. If you aren't a builder and don't have the time to devote to a large job, try to use the bedding you already have. Place the cardboard cutouts or plywood at the bed and add just a small paint to finish your work. Or contact a builder in your area and get an estimate of what it will cost to construct the Star Wars bed.
If you're trying to find an entire star wars bedding full experience, you might choose to go beyond buying Star Wars mattress to your bedroom and receive Star Wars bedding. This is quite the best way to imagine you researching your own way through the remote galaxies. If your little one is a Star Wars fan, you're going to be pleased to know there is a bed available in the majority of home made stores with a Star Wars theme. This bed is a mattress with kid rails to prevent children from falling out from the evening. The steel framework sitting low to the floor, also can be utilized with your baby's mattress.
If you're a programmer, consider making beds in-on your own. At-at is your all-terrain armoured vehicle that looks like a creature. To create this specific tool, start with building a very simple Loft style bed. If you're knowledgeable about this CAD image and have applications available for you, you can actually print a scale model for easier production. If not, then try to work. Start with the cardboard template and keep looking until you receive the appropriate dimensions. Once you're happy with your piece, maintain it on your plywood. When you've got electrical experience, consider adding a few flashing lights, then button and changing the work to full effect. This project requires some heavy tools such as generators, electric sanders, screw gun, and a great deal of workout space. The full mattress may take a few weeks depending on the length of time you've got to work on it daily.
Star wars bedding queen size children,


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Gollum, Ents, MASSIVE At GDC Conference
TORn Staffer Darth Caesar ran across this amazing IGN Filmforce report from the annual Gaming Development Conference. See the evolution of Gollum, the Ents, and MASSIVE in this incredible series of video features. Go. There. Now. Run! [More]Posted in Old Main News on March 11, 2003 by Demosthenes


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Performance and 0-60 time
There are multiple versions in the Model S lineup, badged Standard Range, Long Range and Performance with the Standard model using a 75kWh battery and the other two models using a 100kWh battery. Essentially, the higher the number, the longer the Tesla's range and better the performance.
Subscribe to evo magazine
The slowest Model S, the Standard Range, can still hit 60mph from a standstill in 4.0sec, a more than respectable time for a big saloon car. The rest of the range gets quicker still, with the Long Range model taking 3.7sec for the 0-60mph sprint and the Performance covering the same increment in 3.0sec dead. Engage the £8700 Ludicrous Mode – an almost secret setting in the infotainment system that triggers a warp speed-style animation to appear before it's engaged – and the top-spec Model S will accelerate from 0-60mph in 2.4sec.
Tesla's CEO, Elon Musk, wanted to create a car whose performance could match that of the McLaren F1 – a car Musk used to own in his earlier entrepreneurial days. Not only has he achieved that, but the Model S has rather trounced the F1's oft-quoted 0-60mph time of 3.2sec, going a full 0.8sec quicker.
The acceleration the Model S is capable of is most astonishing at low speeds, and particularly off the line where it shoots off without any fuss or wheelspin, just a significant jolt forwards. It loses some of its impact while on the move, but the Model S can still pile on the speed at a respectable rate on to a limited top speed of 155mph for the entire range.


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Retirement Distribution Planning
Better Retirement Planning for Physicians & Their Families
Submit the form below to learn more about how Physicians Thrive can provide additional value to what you're already doing.
*For internal use only. Physicians Thrive will never release your information to another entity or organization for any reason.
See Your Planning Pay Off
Our approach is to ensure you're able to retire the way you want and while avoid unexpected stessors. Our process will measure your current level of financial confidence and includes methods to reduce risks while investing. We will care for you and your accounts now through retirement, helping you invest smart and prevent unforeseen expenses.
Physicians Thrive is mindful that at the heart of every concern about retirement is "will we be okay?". Our retirement security projection was created to address this fear and help you move forward with confidence.
Why Choose Physicians Thrive?
Physicians Thrive has decades of experience helping doctors get a plan they are confident in.
Your advisor won't make blanket recommendations based solely on your status as a physician. Get a plans that is personalized to each client's unique financial situation, goals, and level of risk tolerance.
With all the options out there, our approach what seems complex simply. You define your goals and we help you reach them.
Our advisors find the best opportunities and options for you to have the highest probability of reaching your goals while requiring the lowest amount of funding.
Get Started Today
Each physician is unique and so are their goals. Case by case, we strive to understand each individuals situation.


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RARE Vintage Country French Black & White Resting Bull / Cow Figurine Planter Occupied Japan
Brand Nancy's Daily Dish
This product is unavailable
I bought a pair of vintage bull and cow planters a few years ago which I guess is now a collection since as the saying goes "I've got 3 or more". Because my customers ask for these when I use them in tablescapes that I share on my blog I've decided to pick up duplicates when I see them, which isn't very often, and offer them on my website. So, here we have one of my favorite bull figurines in a planter. It is so realistic looking. This is a vintage piece made in Occupied Japan and still retains the mark on the bottom. It is a very difficult to find piece! These are great for fresh flowers, greens, silk plants or misc. kitchen items. Perfect in French or English country decor!
Made in Occupied Japan
Measures: appx 7 1/2" x 5"
Condition: One nick to one of the horns, no other chips, cracks or damage


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Halloween Scarecrow Character offers 112 awesome poses to inspire your projects. This charming toon character will surely liven up any of your projects and catch the eye of your viewers. Continue reading
The Mummy Cartoon Character is the right choice for you if you want to impress your clients. It is great for your Halloween themed projects. Continue reading
Cartoon character featuring neat and smart look. Our cartoon character set comes in 96 great poses and moods. Get our highly detailed set and use it for your business projects. Continue reading
Smart businessman cartoon character designed in 96 awesome poses that will help your for a wide range of projects. If you looking for smart and friendly character, this one will surely come in handy. Continue reading
The Pumpkin Head Cartoon Character coming with a huge variety of 112 poses will fit your Halloween project and make it even cooler. Continue reading
African-American Cartoon Businessman dressed in formal. We have designed the male cartoon character in 112 great poses and filled all of them with energy and life. Continue reading
Cowboy kid cartoon character made in 112 poses and moods, perfect for the upcoming Halloween holiday. Our set here could be used on any kind of design related projects, no matter if they are personal or commercial. Continue reading
Frankenstein Cartoon Character comes with 112 different poses and moods to breathe life into your Halloween ideas. Continue reading
Today we present our newest business guy cartoon character set – a corporate guy designed in 63 different postures and moods. We did our best to create visually appealing character set that will impress your customers. All poses are filled with energy and enthusiasm, designed with perfection in every detail. Our business guy really looks self-confident and is ready to … Continue reading
Vampire kid cartoon character made in 112 diverse poses. This charming toon character will surely liven up any of your projects and catch the eye of many visitors. Continue reading


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2011 Jaguar XKR
Vehicle has Sold
Vehicle has Sold
Vehicle Specifications:
- CERTIFIED PRE-OWNED VEHICLE with ONE YEAR COMPREHENSIVE WARRANTY from your date of purchase
- No Accident Claims
- Local, Vancouver Vehicle
- Exceptionally Low Kilometres!
- Bluetooth for Hands Free Calling
- 7'' Touch Screen Navigation
- Back Up Camera
- Bowers & Wilkins Premium Sound System
- Siruis Satellite Radio
- Luxury Seating Package
- Super Performance Brakes
- 20" Alloy Wheels
- Heated Front windshield
- Heated and Ventilated Leather Seats
- 16 Way Power Front Seats
- Front and Rear Parking Sensors
- Cruise Control
- Adaptive Dynamics with active Damping System
- Bi-Xenon Headlights with Automatic Lighting Feature
"Why do the British drink warm beer?" They'll tell you that's the proper way to enjoy a pint of Ale, although the age-old car guy joke has always been that Lucas Electrical (aka, the Prince of Darkness) makes their fridges, too. As with all matters of taste, we'll leave that to personal experience. Of course, newer British cars have long abandoned Lucas electrical systems for more reliable German Bosch parts, but the stories remain: "get home before dark, " as Lucas was the inventor of the "self dimming headlamp, " and "the patent holder for the short circuit."
The 2011 Jaguar XKR equipped with a modern Bosch electrical system, however, leaves nothing to be desired. Wrapped in a timeless all aluminum wrapper, this car exudes confidence and sex appeal. Think of it as the automotive variant of Idris Elba: muscular, brooding and reliably unbothered.
Stomp on the go-go pedal and be prepared to hold on: with 510HP bursting to life out of its 5.0L V8 supercharged engine, this car is not for the meek. 0-100 km/hr comes up in an alarmingly quick 4.6 seconds, despite some electronically limited slip from its bespoke 20" wheels out of the gate. Go into a corner hard, and the car rewards with surefooted grip and a communicative, lively steering feel.
But this car isn't all about driving fast and furiously around corners and mountain passes. Rather, it's GT-car proportions, leather interior and high fidelity sound system make it an equally enjoyable cocoon from urban chaos, as it is enjoyable on a long stretch of sun filled highway with the windows down, and Meridian Sound system blaring CCR's "Fortunate Son."
Like Mr. Elba, this car's magic lies in its mysterious good looks and quiet character. Just be prepared for the occasional plot twist.
Out of Town Purchases + Delivery:
We have customers across Canada, the US, and through parts of Western Europe. As much as we want to sell cars in British Columbia, we recognize that our brand and business has earned its reputation internationally.
In Canada, every province has different taxation rules and we can accommodate them all. We can help you secure worry free shipping to your door, and assist you with vehicle registration once your car arrives.
You have a number of choices available to you:
The first step is to contact us. We always have our professional photographs, our inspection reports, lien checks, service records, CarProof, and CarFax Reports available to you. We're happy to refer you to previous out of town customers so you can get a reliable third party account of their experience with us, to give you peace of mind when purchasing a car sight-unseen.
Should you prefer to see, touch, and drive your vehicle prior to purchase, we will pick you up at Vancouver International Airport (YVR). From there, you can choose safe, insured door to door delivery in enclosed transporters that guarantee the arrival of your purchase in the same condition that it left our showroom.
Or, should you opt for an exceptional drive home, we will happily pay up to $250 toward your overnight stay en route. Please contact us if you have questions or need further information on out of province or international purchases, shipping, and/or sales tax exemptions.
The Fine Print:
Smart people always read the fine print, and you're smart, so here it is:
- all vehicles subject to prior sale
- all prices subject to applicable taxes and levies
- *Lease payments are quoted on approved credit, and are subject to change from month to month. Applicable taxes, lease acquisition fee and PPSA registration fees additional. Contact us for details or to tailor a quote to your preference. We have terms up to 60 months with as little as $0 down available on most vehicles.
- while we make every effort to ensure our website is correct, sometimes mistakes can, and do happen. Please contact us for exact vehicle options, history, mileage and details. Better still: come in and experience the car for yourself, to confirm it's right for you. E.+O.E.


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"Everything that rises must converge," the great American novelist Flannery O'Connor observed a half-century ago.
We're clearly seeing the convergence of at least four trends, two old and two new, and according to newly released findings, marketers may not be prepared for what may prove to shake their world. In fact, a survey of marketing professionals conducted on behalf of BrightEdge, an SEO and content marketing firm, found substantial confusion at what may be a critical moment in the rapidly pacing cycle of digital consumption.
The four trends that are about to come together in dramatically new fashion are:
- Search-engine optimization
- Content marketing
- Artificial intelligence
- Voice search
SEO and content making positive headway.
We're already seeing the convergence of SEO and content marketing, two disciplines that, for much of the digital lifetime, ran in uncoordinated parallel.
Smart marketers are getting more skillful at content strategies that understand customer intent and then target content accordingly. In the aforementioned survey, 97 percent of respondents agree SEO and content marketing are converging, or have already converged. The same survey found organic search is the number one priority — a smart focus considering organic drives 51 percent of all website traffic.
Effective use of these strategies demands some deep thinking about the steps consumers take during their purchase-making decisions — and the words that are likely to move them at each stage of their journey. Otherwise, effort and resources will be lost on content that is inaccessible.
Layering in new technologies.
The wider adoption of artificial intelligence supercharges marketers' ability to fine-tune SEO strategies. One of the biggest differences is capability of AI to put consumers' searches into context.
When a consumer searches for "Tahoe" today, he might want a Chevrolet SUV or a winter ski vacation. AI puts that search into the context of the user's past searches — pickups or snowboards — and makes some educated guesses about the user's intentions.
Read the rest of the article at entrepreneur.com.


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I'm so excited for upcoming SBS Wed-Thurs drama Suspicious Partner, regardless of the leading lady casting carousel and the litany of title changes. I never got invested in currently airing sageuk Saimdang: Light's Diary and judging from the steady around 10% ratings neither did the audience despite the big name return of Lee Young Ae from a decade long acting hiatus. A quirky rom-com sounds perfect for the spring season and it doesn't hurt to see leads Ji Chang Wook and Nam Ji Hyun radiate cuteness together at the script reading for the drama. I don't see any age difference and frankly he's the same age as Seo In Guk who had amazing chemistry with Nam Ji Hyun in their last rom-com Shopping King Louie. I pray this one has as engaging a story will equally caring characters, please don't waste their talents and also give Ji Chang Wook a nice send-off to military service later this year. Continue reading
Tag Archives: Nara
It's all come together now for Kim Eun Sook's upcoming tvN fantasy drama Goblin, a male lead dominated production that continues Kim Eun Sook's trend of finding the most charismatic male leads around. Starring Gong Yoo and Lee Dong Wook, … Continue reading