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Kentucky Farm Bureau Insurance Company Reviews
Kentucky Farm Bureau Insurance Company is an Independent Organization. Kentucky Farm Bureau is Popular in Providing their services to Farm Families.
Kentucky Farm Bureau is Dedicated as Backbone in the Agriculture department. The KY Farm Bureau Identifies the Problems in Agriculture and takes Action on that and Develop Solutions of that Problems.
Their Services in Agriculture will Improve the Net Income of the Farms. By taking their Services the Farm Families can accomplish better financial opportunities and enhance the quality of their lives.
The Headquarters of Kentucky Farm Bureau is situated in 9201 Bunsen Parkway Louisville, Kentucky, 40250, United States of America.
Before Getting Insurance Premium Also Read about USAA Insurance Company
History of Kentucky Farm Bureau
Almost 100 years Old Organization Kentucky Farm Bureau Company was founded in November 1919 at a meeting in Louisville. The Organization is Providing its Services For more than 75 years and now Know as the "Voice of Kentucky Agriculture."
The KY Farm Bureau has more than 500,000 Registered Members. These Registered Members brings the Organization in the top three Farm Bureaus for the last Ten Years.
The organization's remaining control has been the result of numerous factors, but the foremost imperative is the nature of Farm Bureau's administration structure. Control of Farm Bureau, whether at the district, state or at the national level, is invested within the hands of agrarian makers, comprising the grass-roots authority.
Services of Kentucky Farm Bureau
Kentucky Farm Bureau Company has built a well-known reputation to the Farm Families. In other words, we can say that KY Farm Bureau became a Leader in Agriculture in front of its Members. The informatory Products and Member Service Programs are also very successful.
KY Farm Bureau is also producing the Half-Hour Television Program Bluegrass & Backroads. The Production of the Program was started in 2003. The Program can be watched on RFD-TV throughout Nationwide.
The TV Program provides information about Public Affairs, Kentucky Farm Bureau Insurance and other Member Services, Safety, Health and Wellness, Women's Leadership Activities, Estate Planning, Theft Reward, Scholarships, Commodity Market Information, Certified Roadside Farm Markets and Young Farmers.
Insurance Coverage offered by Kentucky Farm Bureau
If you are interested in getting Insurance Policy from the Kentucky Farm Bureau Company and want to know the types of Insurance Policies then there are following types of Insurance Coverage offered by KY Farm Bureau.
Note: These Insurance Coverage are offered for Car Insurance Policy.
Personal Injury Protection: If you are the policyholder of the Kentucky Farm Bureau. They will pay all the medical bills in case of getting some injuries.
Bodily Injury Liability: If you have got a Car Insurance Policy from KY Farm Bureau. Then they will Protect you in getting Some Injuries while driving.
Property Damage Liability: If you got an accident and someone's property is damaged. They will cover this damage for you.
Collision Damage: If your car is damaged in an accident and you are a policyholder then Farm Bureau will pay the Car Repair Costs if your car is damaged in Collision.
Comprehensive Damage: A policyholder can get their cars repair which is damaged from weather, debris and other non-collision related incidents.
Uninsured and Underinsured Motorists: If you got an injury by an uninsured Driver then you can get paid medical bills from the Farm Bureau.
Benefits offered by Kentucky Farm Bureau
Rental and transportation reimbursement: If your car is repaired from any other Place. You can get Covered for you have used to get around.
Medical payment coverage: If you are injured in Collision. All your medical bills can be paid from the Kentucky Farm Bureau.
Towing and labour cost coverage: You can pay less amount when your car got a repair from Kentucky Farm Bureau. You have not to pay Labour cost while getting a repair.
Discounts Offered by Kentucky Farm Bureau
There is a huge number of Premium Discounts offered by KY Farm Bureau to is customers. If you are also its customer you may qualify for the discounts.
So, the List of Discounts is given bellow.
Auto Safety Device Discount: You can get a discount on Antitheft devices, Antilock brakes, and other safety equipment.
Active Property Discount: When you have Homeowner Insurance, farm owner or mobile homeowner insurance with KY Farm Bureau. You can qualify for a discount on getting auto insurance Policy.
Driver's Training Discount: If you are a guy younger than 21 or Single Driver younger than 25 you can get a discount on completing a safe driving course.
Safety Driving Course Discounts: Drivers who have the age of more 55 years can claim a discount by completing a safety driving course. Drivers belong or Retired from US military can also get the discount by showing that they have also completed the Safety Driving Course from Military.
Good Student Discount: If you are completing a driving course you can also get a discount as your Reward if you got the good Grades.
Multivehicle Discounts: If you have more than one vehicles then you can get a discount on your premiums by getting insured by KY Farm Bureau.
Safe-Driver Discount: If you spend three, five or eight accident-free years then you will also get a discount on your premiums.
Umbrella Discount: Get Umbrella and auto insurance Policy from KFB and get a discount on both.
Pros of Kentucky Farm Bureau
Number of Discounts: They have a bunch of discounts on their Premiums. You can also qualify and can get discount from Kentucky Farm Bureau Insurance Company .
Agents in 120 Counties: They have a huge number of agents available in 120 counties. You can get any type of information from them through Phone Call or by met them Personally.
After-hours claims Reporting: If you have got any type of accident or facing any trouble after business hours. You can Report them after working hours through a dedicated hotline.
Member Benefits: Members of this Policy Provider can get access to the discounts and deals on auto, travel, health, home and farm insurance policies.
Cons of Kentucky Farm Bureau car insurance
Only Available in Kentucky: The Insurance Company Provides their Services only in Kentucky. People from out Kentucky can not apply for their Services.
Limited Online Support: If you are Looking for the Information for their Services. You will only get basic information about them. There is also no option for online chat or call. You can only get
all type of information from their agents.
Agent Support Only: As we described before that there is no online Support. If you have decided to get a policy then the only agent can support you in this Purpose.
The Process to Apply for Kentucky Farm Bureau
If you have decided to apply for Insurance Policy with Kentucky Farm Bureau Insurance Company then you have to follow the Steps, Explained Bellow. You can get a quote over the phone as well.
- Go to the Official Website of Kentucky Farm Bureau Insurance Company .
- Select on the Option Get a Quote.
- Fill out the Given form with Required things Such as Name and Address and then click the Next
- Select a Preferred Agent and then hit the Next
- Click on the Icons for the Insurance Products you are interested in.
- Fill out the form with your Email Address, Your Phone Number and the Best Time to Contact with You.
- Hit the FinishAll Process is done and now wait for Some Time to Get Agent Back to You.
The Process to make a claim with Kentucky Farm Bureau
If you are in Trouble and want to Report a claim then you can apply the following two methods according to the time.
- If you are filling a Claim Report between 9:00 A.M to 4:30 P.M through Monday to Friday. Then Call them in their Local Office.
- If you have decided to fill the claim report on non-business hours then you can them on their dedicated hotline 866-532-2524.
At the Time of Filing the Report, You have Required the Following Information
- Your Insurance Policy Number.
- Names and Phone Numbers of Eyewitnesses and Included Passengers included in the Incident.
- Time and Date of the Incident
- Details about Vehicle and Property involved
- Location of the Accident
- Police Report of that Accident Number
- Description of Damages and Injuries
Infographic about Kentucky Farm Bureau
This Infographic defines the Services of Kentucky farm bureau insurance company
Contact Details of Kentucky Farm Bureau
If you are interested in getting Policy or have any Complaint then there are multiple ways to contact the Kentucky Farm Bureau.
|Official Website
|Kentucky Farm Bureau
|Kentucky Farm Bureau
If you Lives in Kentucky and Looking to Get an Insurance Policy. Then Kentucky Farm Bureau Insurance Company may have something extra according to your needs and imagination. You can get Some Extra Benefits from the Top Rated Insurance Company. So, it's up to you how you can take benefits from the offers and discounts given by KY Farm Bureau on getting Insurance Policy.


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Kapoor sisters Kareena and Karisma celebrated their mother and yesteryear actress Babita Kapoor's birthday on Sunday evening.
Though Babita's birthday is on 20 April, the Kapoor clan celebrated it the previous day. Son-in-law Saif Ali Khan also joined the bash, along with Babita's husband Randhir Kapoor, Armaan Kapoor and other family members.
The celeb family was snapped when they were coming out of the venue. All of them smiled and posed for the shutterbugs.
Both the daughters were looking glamorous in their perfect outfit for their mom's birthday party. Even Saif was in his royal look.
Bebo was recently spotted in Delhi with Sonam Kapoor and Arjun Kapoor at India TV Yuva Awards. The "Bajrangi Bhaijaan" actress arrived in Mumbai on Sunday itself and attended her mother's birthday party in the evening.
See Photos of Kareena and Arjun Kapoor at Mumbai Airport
Check out the above slideshow of Babita Kapoor's birthday bash.


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STOP FEELING SORRY FOR ME…and stop feeling sorry for yourself. Stop putting your hand to the plow and looking behind you. This your life and your greatest results will come from your consistent choices and identity.
Love your CHOICES. Never feel sorry for yourself. … Here's how.
For a long time I was in conflict between my desire for fitness and my desire to satisfy my taste buds. I believed i had to GIVE UP something to get what i wanted but i was only willing to give it up 'short term'. I wore my sad face when I struggled to lose weight with self denial and mysteriously believed when i returned to a normal (crappy and unhealthy) diet, it was "normal".
Really? WHY should I feel sorry for myself when I'm doing the things that result in success? That's Crazy!
As a health coach, now i realize the power of choice and how much I love the decisions that have allowed me to be healthy, lean and feel much younger than i did a decade ago. But i see so many people struggle with this UPSIDE DOWN perspective that they are SUFFERING if they give their body Optimal Fuel to perform in an Optimal way for an Optimal Life! (did i overdo it there?)
STOP FEELING SORRY FOR ME. And STOP FEELING SORRY FOR YOURSELF.
You have been given the power to choose and that means you have the power to choose your destiny, not be dictated to by some random bowl of Pasta or Pan of Pizza. SURE, you can have some, but when it runs your life… you're going to run out of life.
I am the master of my destiny and my choices. So are you. I choose ME, especially when it comes to optimal health.
Isn't it time to celebrate the power of Choice. CHOOSE YOURSELF and never be ruled by the deception of momentary fixes. You deserve it.
For more on the conversation, follow me on the web and on Social media. I'd love to talk to you about health coaching and getting results, no matter what your plan is. We have a powerful strategy and a powerful community walking together to Optimal Health.
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Turning a protein kinase on or off from a single allosteric site via disulfide trapping
Contributed by James A. Wells, February 10, 2011 (sent for review September 28, 2010)
There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)—the PDK1-interacting-fragment (PIF) pocket—is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (< 300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.
Allosteric sites on protein kinases are difficult to find, characterize, and target with small molecules. Whereas the development of active site inhibitors has benefited greatly from the existence of a natural small molecule substrate, ATP, there are virtually no natural small molecule starting points from which allosteric kinase modulators can be built. Thus, most allosteric modulators have been discovered serendipitously, generally through high-throughput screening (HTS), which is inherently inefficient. Confirming the binding sites and mechanisms of action of allosteric modulators also tends to be more laborious relative to ATP-mimetic inhibitors. Prior to initiating HTS campaigns, it would be beneficial to have a simple method that permits directed interrogation of kinase allosteric sites with small compounds and facile assessment of the effects of binding on protein structure and function.
We envisioned that a site-directed approach developed previously by our laboratory, disulfide trapping (or tethering), would allow more straightforward characterization and interrogation of allosteric sites on protein kinases with small molecules. Disulfide trapping involves screening disulfide-containing compounds for their ability to form a mixed disulfide with a natural or engineered cysteine residue near a site of interest on a protein target (1, 2). Because the approach is site-directed, disulfide trapping has proven valuable as a tool to validate known, suspected, and orphan allosteric sites on protein surfaces as small molecule targets (3–6), but has not yet been applied to kinase allosteric sites.
Here, we use disulfide trapping to target an allosteric site on the surface of 3-phosphoinositide-dependent kinase 1 (PDK1)—the so-called PDK1-interacting fragment (PIF) pocket. PDK1 is a member of the AGC family of Ser/Thr kinases, which includes protein kinase A (PKA), B (PKB/Akt), and C (PKC) isozymes. In PKA, PKB, and PKC the site analogous to the PIF pocket binds to a specific hydrophobic motif (HM) at the C terminus of these kinases (7). In PDK1, which lacks its own HM segment, the PIF pocket serves a dual purpose: It recruits downstream substrate kinases that possess an HM segment (e.g., PKC, S6K, RSK, and others) and, upon HM binding, allosterically stimulates PDK1 activity (8–11). Short peptides (12–25 residues) derived from the HM of several kinases (PIFtides) can activate PDK1 by about four- to sevenfold, although their mode of interaction with PDK1 has not yet been established (8, 9). Recently, small molecules that mimic PIFtides and similarly activate PDK1 by binding the PIF pocket have been discovered and characterized structurally and biochemically (12).
We used disulfide trapping to (i) better elucidate how a PIFtide binds to and activates PDK1, (ii) identify new small molecule fragments targeting the PIF pocket, and (iii) begin to characterize their allosteric mechanism. Most remarkably, some of the disulfide-trapped fragments activate and others inhibit the kinase. Biochemical and X-ray structural studies provided insight into the binding modes of allosteric modulators and protein conformational changes that may propagate their allosteric effects. These studies provide a framework for systematically assessing functional and structural effects of small molecules directed at specific allosteric sites on protein kinases.
Trapping of Peptides at the PIF Pocket on PDK1.
Our first goal was to use disulfide trapping to better characterize the binding of PIFtides to PDK1. Because no crystal structures of PIFtide/PDK1 complexes have been reported, we have generated an energy-minimized homology model based upon the known structure of a chimeric Akt construct, in which the endogenous C-terminal HM was replaced by the HM of the kinase PRK2 (13) (Fig. S1A).
To test our model, we prepared a panel of recombinant PDK1 Cys mutants in which residues at six positions around the PIF pocket were mutated individually to a cysteine (K115C, I119C, V124C, R131C, T148C, and Q150C). All of these Cys mutants displayed comparable catalytic activity, but were somewhat less active than the wild-type enzyme (by approximately 20–50%; Fig. S2). We next synthesized a panel of 11 PIFtide variants derived from the HM of PRK2 in which a disulfide-capped cysteine replaced residues along the sequence (Fig. 1A). We allowed each of the six Cys-mutant kinases (at 1 μM) to form a disulfide adduct with each of the 11 Cys-PIFtides (at 100 μM). The concentration of β-mercaptoethanol (β-ME) was varied (100 μM or 1 mM) to adjust for differences in intrinsic reactivity toward disulfides of the introduced cysteines on PDK1, as gauged by promiscuity in binding to the library of Cys-PIFtides. The level of conjugation was determined by mass spectrometry.
The Cys-PIFtides showed pronounced selectivity among the six PDK1 Cys mutants (Fig. 1B). Furthermore, the extent of conjugation tracked well with the predicted proximity between the cysteines on the protein and the peptide according to our calculated model for the PIFtide/PDK1 complex. For example, PDK1K115C conjugated extensively to the Cys at position 3 in PIFtideQ3C, and the extent of conjugation to other PIFtides tapered off with distance from residue 115 in PDK1. Similar effects were seen with conjugation to Cys at four other positions: 119, 124, 131, and 148. One of the PDK1 mutants, PDK1Q150C, was significantly more promiscuous in its reactivity although this mutant did show strongest conjugation to the residue on the PIFtide (D10C) nearest to it in our model.
We tested if the conjugation data could be used to further refine our initial homology model. We performed a conformational search using a conservative restraint of 7.0 Å on the Cβ–Cβ distances between PIFtide residues and residues in PDK1 that showed the greatest conjugation in disulfide-trapping experiments. We followed up with unrestrained molecular dynamics (MD) simulations with explicit aqueous solvent. The PIFtide remained bound to the pocket throughout the simulations and did not deviate greatly in conformation relative to the initial constrained model. By contrast, models derived from MD simulations on an initially unrestrained complex showed significantly greater heterogeneity and sampled conformations very different from that of the starting structure (Fig. S1 and Movie S1).
We next assessed the effects of disulfide-trapped PIFtides on the catalytic activity of their associated PDK1 Cys mutants (Fig. 2). These assays were performed with peptides and PDK1 mutants for which we could achieve stoichiometric conjugation at the lowest β-ME concentration tested (100 μM). Kinase activities (initial rates) were measured with a standard assay using a peptide substrate taken from the activation loop of Akt and radiolabeled ATP.
All of the PDK1 Cys mutants tested had at least one Cys-PIFtide that when conjugated increased kinase activity relative to unconjugated PDK1 (Fig. 2A). The catalytic activity of the most activated complex, PIFtideD10C/PDK1Q150C (5.2-fold activation), even exceeded that which we measured for the noncovalent complex between the parent PIFtide and wild-type PDK1 (3.9-fold activation at saturation). Interestingly, although several PIFtide disulfides could be conjugated stoichiometrically to PDK1Q150C (Fig. 1B), PIFtideD10C was clearly more effective than the others at activating this kinase (Fig. 2B). The average errors in activation or inhibition potency did not exceed 15% of the values shown (see Experimental Procedures).
Trapping of Small Molecules at the PIF Pocket.
We next evaluated whether small molecule disulfides would mimic effects seen for the larger Cys-PIFtides (molecular mass of approximately 2,000 Da). Thus, we screened each of the six PDK1 Cys mutants against a library of 480 disulfide-bearing fragments (molecular mass 200–500) that was synthesized in the Small Molecule Discovery Center at University of California, San Francisco. These compounds represent a diversity of scaffolds common in drugs and drug candidates (e.g., heterocycles, piperazines, etc.) and most obey the "Rule of 3," which was established to judge fragments as suitable starting points for drug discovery efforts (mass < 300 Da, predicted cLogP ≤ 3, number of hydrogen-bond donors/acceptors ≤ 3) (14). Preliminary experiments revealed that the disulfide fragments reacted much more readily with the PDK1 Cys mutants than did the Cys-PIFtides; at 100 μM β-ME and 100 μM compound, most small molecule disulfides reacted quantitatively with every Cys mutant. Therefore, in screening disulfide fragments for binding, we increased the assay stringency by raising the concentration of β-ME to 10 mM.
Under our assay conditions, the hit rate for disulfide fragment binding varied from 1.3% to 13.8% for the six PDK1 Cys mutants (using 20% conjugation to define a hit). The least hit-rich PDK1 mutant, PDK1I119C, trapped only six compounds, whereas the most hit-rich mutant, PDK1K115C, trapped 66 compounds. Interestingly, the mutants that were the most promiscuous in binding to small molecules (K115C, R131C, and T148C) were the most selective in binding to Cys-PIFtides (Fig. 1B). About 3% of the disulfide fragments in the library (15 out of 480) were trapped to > 50% conjugation to one and only one of the six PDK1 Cys mutants. The wild-type PDK1 catalytic domain has four endogenous cysteines, which were retained in our mutant constructs. Nevertheless, none of the fragment disulfide hits conjugated to wild-type PDK1, indicating specific binding to the Cys incorporated at the PIF pocket.
Whereas Cys-PIFtides activated PDK1 mutants, the disulfide fragment hits comprised both activators and inhibitors, as well as several inert ligands that neither activated nor inhibited the enzyme (Fig. 3 A and B and Fig. S3). Every PDK1 Cys mutant produced all three outcomes that varied depending on the fragment bound. Some mutants (e.g., K115C and Q150C) seemed to trap inhibitors or activators preferentially, although the sample size underlying these trends is small. The mutants showing the greatest range in activity on stoichiometrically trapping small molecule disulfides were PDK1R131C and PDK1T148C where activity varied about 14-fold from the most inhibited to the most activated complex. The effects of compound concentration on percent conjugation (Fig. 3C) and kinase activity (Fig. 3D) were compared for the most activating and inhibiting fragments targeting PDK1T148C—2A2 and 1F8, respectively. The concentrations required for 50% conjugation to this mutant (DR50 values) were 5.6 μM for 2A2 and 4.6 μM for 1F8 (at 1 mM β-ME). These values tracked closely the concentrations required for half-maximal effect on catalytic activity (EC50 values)—7.1 and 7.2 μM for 2A2 and 1F8, respectively. None of the most activating or inhibiting disulfide fragments had a significant impact on the activity of wild-type PDK1 and all were approximately equally effective toward mutants in the absence and presence of the detergent CHAPS, which is known to disperse small molecule aggregates that can lead to spurious enzyme activation or inhibition (15–17) (Fig. 3B and Fig. S4). Overall, these results indicate that specific disulfide trapping of compounds at the PIF pocket is critical for the functional effects observed.
Optimizing a Disulfide Fragment Activator.
To assess relationships between structure and activity of disulfide activators, we synthesized and tested 30 analogs of 2A2 in which the N-chlorophenylpiperazine moiety was replaced with various N-substituted piperazines. Compounds were then assayed for binding to and activation of PDK1T148C (Table S1). Only N-phenyl and N-benzyl piperazines conjugated detectably to this mutant, whereas similar N-acyl and N-benzoyl derivatives did not (at 100 μM compound, 10 mM β-ME). Substitution of the aryl ring within these derivatives significantly affected both conjugation strength and activation potency and these properties did not seem to be correlated. The best compound of the series ("JS30"; Fig. 3B) activated PDK1T148C by 6.3-fold, representing a significant improvement over 2A2 (3.9-fold).
Biochemical Characterization of Allosteric PDK1 Modulators.
To explore the mechanism behind activation and inhibition of PDK1 by PIF pocket ligands, we first wanted to assess whether binding at the PIF pocket is coupled to substrate binding at the active site. The weak affinity of the Akt-derived substrate peptide for PDK1 (Km approximately 10 mM) precluded measurement of Michaelis–Menten parameters (8). Thus, we chose to assess interplay between the ATP pocket and the PIF pocket using disulfide trapping. Dose-response curves were measured for binding of the best disulfide fragment activator (JS30) and the best inhibitor (1F8) to PDK1T148C in the presence and in the absence of a saturating concentration of staurosporine (100 μM), a tightly binding, ATP-competitive inhibitor of PDK1 (Fig. 4A). These curves showed only minimal effects of staurosporine on the efficiency of disulfide-trapping for either JS30 or 1F8.
We next probed whether the allosteric PDK1 modulators affected local or global protein stability. Park and Marqusee have shown that susceptibility of a protein/ligand complex to proteolysis by a nonspecific protease (e.g., thermolysin) is inversely proportional to the energetic stability of the complex (18). We compared thermolysin-catalyzed degradation of PDK1T148C alone and bound stoichiometrically to one of our best allosteric small molecule modulators (JS30 or 1F8) or an inert allosteric ligand (1H9) as visualized by SDS-PAGE (Fig. 4B). All of these ligands stabilized PDK1T148C to degradation; the half-life of the protein was 1.9 min in the absence of these compounds and 6.7–9.5 min in their presence, representing 3.5- to 5-fold stabilization. The half-life of wild-type PDK1 (2.5 min) was, by contrast, insignificantly affected by the disulfide fragments (Fig. S5).
We also performed hydrogen-deuterium (HD) exchange experiments to assess stabilization of PDK1T148C bound to allosteric modulators (Fig. S6). Upon stabilization, one expects hydrogen-bonding interactions involving backbone amide protons to become stronger, limiting exchange with deuterium from D2O (19). We exposed unliganded PDK1T148C and PDK1T148C bound stoichiometrically to JS30, 1F8, or 1H9 in H2O to D2O and monitored deuterium incorporation by mass spectrometry. The mass of the protein alone shifted upward by 76 Da upon exposure to D2O for 1 min, whereas the mass shift was +53, +52, and +51 Da for PDK1T148C bound to JS30, 1F8, or 1H9, respectively. Thus, 23–25 amides, or approximately 30% of the total number of exchangeable amides, were protected from exchange in the bound versus unbound protein.
Structural Characterization of Allosteric PDK1 Modulators.
To gain deeper insight into the binding mode and possible mechanism behind allosteric modulation of PDK1 by PIF pocket ligands, we solved high-resolution X-ray crystal structures of three complexes, two between PDK1T148C and a disulfide fragment activator, 2A2 or JS30, and one between this mutant and a disulfide inhibitor, 1F8 (to 2.0, 2.1, and 2.2 Å resolution, respectively; Table S2). Both activated complexes were crystallized in the presence of the known ATP-competitive ligand bisindolylmaleimide-II (BIM-II), whereas the inhibited complex was crystallized in the absence of an ATP-site ligand.
Continuous electron density could be traced in all three disulfide-bound PDK1T148C structures from Cys148 to the appended fragment, 2A2, JS30, or 1F8, allowing unambiguous determination of the position of these ligands in the PIF pocket (Fig. 5 A–C). All three fragments target the same general area in the PIF pocket of PDK1, filling a hydrophobic cavity lined by the side chains of residues Lys115, Ile118, and Ile119 on helix B; Val124, Val127, Thr128, and Arg131 on helix C; and Gln150, Leu155, and Phe157 on β-strand 4. However, the shape of this cavity differs in the three structures due primarily to movements of the B and C helices (see below).
In all three structures, PDK1T148C adopts an active-like conformation overall, as characterized by specific interactions among conserved catalytic residues (Fig. 5D and Fig. S7). For example, salt-bridge interactions are formed between Glu130 on the C helix and Lys111, residues that are critical for positioning ATP for phosphoryl transfer. Salt-bridge interactions are also apparent between phosphorylated Ser241 (pS241) on the activation loop and two key Arg residues, Arg204 of the so-called HRD motif and Arg129 on the C helix, which are responsible for positioning the activation loop to accept a protein/peptide substrate (20).
Despite the similarities among the inhibited and activated PDK1T148C structures, several differences are also apparent (Fig. 5D and Fig. S7). For example, the conformation of the activation segment (residues 223–252) varies. In the two activated structures, this segment is largely disordered and displays poor electron density between residues 232 and 240. A poorly-defined activation loop was also observed for one of the two conformers found in the asymmetric unit of the inhibited 1F8 complex, whereas in the other conformer, the activation segment could be almost completely refined, forming a short α-helical turn between residues 231 and 236. The positions of the B and C helices are also closer to the activation loop and ATP-binding site in the 2A2 and JS30 complexes relative to the inhibited 1F8 complex. Residues on the C helix adopt different conformations in the three structures. For example, in the most activated complex (with JS30) the Tyr126 hydroxyl makes putative H-bonding interactions with the side chains of Asp223 (in the DFG motif) and Glu130 and with the backbone amide of Gly225 (in the DFG motif). In the 2A2 complex, the Tyr126 hydroxyl is positioned slightly further away from these residues and makes a hydrogen bond to a water molecule that instead makes the above contacts with the DFG motif and Glu130. In the inhibited 1F8 complex, the Tyr126 side chain adopts two conformations in the crystal, one in the direction of the active site and one forming an H-bonding interaction with pSer241, but both too far from the DFG motif or Glu130 to form contacts with either.
We mutated Tyr126 in PDK1T148C to Phe to investigate the role of this residue in activation/inhibition by PIF pocket disulfide compounds (Fig. S8). The resulting Y126F/T148C double mutant had approximately twofold lower kinase activity relative to the T148C single mutant. The double-mutant was less well activated by JS30 than was PDK1T148C (5.2- vs. 6.0-fold activation, respectively), but was inhibited slightly better by 1F8 relative to the single mutant (3.8-fold vs. 3.1-fold inhibition).
Unlike conventional screening approaches, disulfide trapping is well suited for hypothesis-driven ligand discovery efforts of the type described here in which one asks whether targeting a specific protein site with small molecules or peptides can produce a desired functional and/or structural effect. Here, we have used the technology to gain a better understanding of how PIF peptides interact with and allosterically activate PDK1 and to probe the PIF site with small molecule fragments.
Molecular modeling simulations provided a structural hypothesis for how PIFtides bind to PDK1 and we subsequently used disulfide trapping in solution to test this hypothesis. Taken together, our conjugation data strongly support a binding orientation of the PRK2-derived PIFtide in the PIF pocket on PDK1 that is similar to that adopted by hydrophobic motifs of AGC kinases bound intramolecularly to their own analogous (HM) pocket (7). Comparison of the catalytic activities of different cross-linked peptide/PDK1 pairs showed that the extent of PDK1 activation is sensitive to PIFtide positioning. For example, several Cys-PIFtide/PDK1 mutant cross-links could be formed between residues that were nearby in our model, but these were nonequivalent in terms of PDK1 activation. That the most activated complex, PDK1Q150C/PIFtideD10C, was more active than the corresponding wild-type (noncovalent) PIFtide/PDK1 complex suggests that the full activation potential of wild-type PDK1 has not been realized in experiments with isolated PIFtides. It is possible that the entire PIF-containing protein (i.e., PRK2) is required for inducing maximal activation of wild-type PDK1.
Screening of a small library of 480 disulfide fragments yielded a number of ligands with diverse structures and functional impacts on PDK1. Each Cys site on PDK1 trapped a distinct panel of disulfide fragments that were selective for each mutant over other mutants or the wild-type enzyme, suggesting specific binding modes in each case. The best disulfide fragment activators from our initial screen were nearly as potent as the best Cys-PIFtide activators; chemical analogs produced even more potent fragment activators (reaching 6.3-fold activation).
Perhaps most surprising was the fact that the fragments, unlike PIFtides, included both PDK1 activators and inhibitors. Both binding and extent of activation or inhibition were highly dependent on chemical structure of the bound fragment and positioning of the fragment in the PIF pocket. The activity of PDK1T148C in complex with small fragments was variable over the largest range (approximately 19-fold), from the most inhibited (in complex with 1F8) to the most activated (in complex with JS30). Although the fragments are only about 1/10th the size of PIFtides, their effects on PDK1 activity were more extreme (i.e., greater activation and inhibition). It is possible that the activation of PDK1 observed by PIFtides represents the net sum of interactions between a number of subsites, some of which can be activating and some inhibiting. Perhaps the small fragments can occupy individual subsites and thereby generate more potent activating or inhibiting effects on PDK1. Previous disulfide-trapping experiments on the C5a receptor, a G-protein coupled receptor (GPCR), produced both agonists and antagonists at the same site (4, 5). It is intriguing that a single allosteric site on a kinase, the PIF pocket on PDK1, can similarly generate a continuum of agonistic and antagonistic effects, by analogy to single ligand binding sites in GPCRs.
Biochemical data and high-resolution crystal structures for activating and inhibiting fragments bound stoichiometrically to PDK1T148C showed that these ligands stabilize the protein globally and induce conformational changes that may account for their effects on activity. In crystallizing our most strongly inhibited complex, 1F8/T148C, in the absence of an ATP-site ligand and the most strongly activated complex, JS30/T148C, in the presence of an ATP-site ligand (BIM-II), we expected to observe widely divergent PDK1 conformations. Surprisingly, the structures of the 1F8/PDK1T148C and JS30/PDK1T148C complexes were quite similar, with all key catalytic residues (e.g., Lys111, Glu130, and Asp223 in the ATP-binding cleft; Arg204 of the HRD motif; and Arg129 on the C helix) poised in canonically "active" conformations (20). However, clear changes were observed in the conformation of the activation loop and the positions of the B and C helices, which move approximately 5 Å away from the ATP-binding site in the most inhibited versus the most activated complex (Movie S2). Additionally, in both activated complexes, the side chain of Tyr126 in the C helix is directed inward toward the ATP-binding site. In complex with JS30, the Tyr126 hydroxyl on PDK1T148C forms a hydrogen bond with the side chains of Asp223 in the DFG motif and Glu130 on the C helix, residues that are strictly conserved and critically important in all kinases for positioning ATP during catalysis (21). This type of interaction has not, to our knowledge, been observed in other kinase structures. In all PDK1 structures solved to date, Tyr126 is positioned outward, forming an H bond to pSer241. In PKA and PKB, the residue corresponding to Tyr126 in PDK1 is a His residue, which forms an electrostatic interaction with an analogous phosphorylated residue on the activation loop. It is possible that the interaction of Tyr126 with the DFG Asp and/or Glu130 in the JS30/PDK1T148C complex plays a role in allosteric activation. Indeed, mutation of Tyr126 to Phe had a modest, but significant negative impact on activation by JS30. Interestingly, this mutation also makes inhibition of PDK1T148C by 1F8 slightly more effective, suggesting that Tyr126 may also destabilize the inhibited state of the kinase. Prior to phosphoryl transfer in protein kinases, a Mg2+ ion bridges the Asp of the DFG motif and the gamma phosphate group of bound ATP (20). It seems unlikely, therefore, that Tyr126 in PDK1 would be able to form a close interaction with the DFG Asp of PDK1 in the Mg2+/ATP-bound state. However, it is possible that Tyr126 assists in release of ADP following phosphoryl transfer, a step which is rate limiting for many kinases.
Comparisons among the biochemical and structural data reported here for disulfide-bound modulators of PDK1 and elsewhere for PDK1, alone and in complex with a known small molecule activator that binds to the PIF pocket, PS48 (12), provide additional clues regarding the mechanism(s) behind allosteric modulation of this kinase. For example, PS48 was reported to stabilize PDK1, but was paradoxically competitive with ATP binding (12). We also observe kinase stabilization by PIF pocket-directed disulfide fragments, but do not observe significant communication with the ATP site by PIF pocket activators, suggesting that this feature is not necessary in all cases for activation. Our disulfide fragments also bind to the PIF pocket in slightly different ways relative to PS48. Like PIFtides, PS48 possesses two aromatic groups that become buried in the hydrophobic PIF pocket and a carboxylate that forms an electrostatic interaction with Arg131 on the C helix (12). Only the hydrophobic components are reproduced in the disulfide activators presented here, yet activation is observed. The carboxylate/Arg131 contact between PIFtides/PS48 and PDK1 may therefore be important for binding to the PIF pocket, but may not be important for activation. Finally, a comparison of the structures here of PDK1T148C bound to inhibitory and activating disulfide fragments and those previously reported of PDK1 absent a PIF pocket ligand (22) and in complex with PS48 (12) reveal a compelling correlation between C-helix position and kinase activity (Fig. 5D and Movie S2). The positioning of the C helix relative to the ATP site for PDK1 bound to various PIF pocket ligands follows the trend, from nearest to farthest, JS30, PS48, 2A2, none (i.e., bound only to an ATP-site ligand), and 1F8, which tracks well with the activities of the corresponding complexes relative to the unliganded proteins: 630%, 400% (12), 390%, 100%, and 32%, respectively. This correlation supports previous hypotheses implicating the C helix in allosteric activation of PDK1 (12), but shows additionally that the C helix may also be positioned to inhibit the kinase. We cannot, however, discount possible effects of crystal packing differences in the various structures as causes for the structural changes we describe.
Many kinases possess a functional allosteric pocket analogous to the PIF pocket on PDK1 that should be targetable using the approach we have described. Within the AGC kinase family, PKA, PKB (Akt), PKC isozymes, RSK, R6K, SGK, PRK1/2, and GRK all have a C-terminal HM segment that docks to a pocket analogous to the PIF pocket and cis activates these kinases (7). In Src, a nonreceptor tyrosine kinase, the analogous pocket is occupied by residues on the SH2-kinase domain linker N-terminal to the catalytic domain, which stabilize an inactive conformation (23). Other kinases are regulated by interactions of separate protein domains with a PIF-like pocket (e.g., TPX2/Aurora A, cyclin/CDKs, and the EGFR/EGFR dimer) (24–26). Based on our success with PDK1, we expect that disulfide trapping will allow us to study and validate such sites in an efficient and systematic fashion while simultaneously providing chemical starting points for developing bioactive kinase modulators.
Experimental Procedures
Peptide Synthesis.
Peptides were synthesized using standard procedures (see SI Text).
Fragment Disulfide Synthesis.
Two equivalents of a commercially available amine or carboxylic acid (Sigma-Aldrich) were reacted with one equivalent of either a succinimidyl ester form of the appropriate carboxylic acid (4,4′-dithiodibutyric acid or 3,3′-dithiopropionic acid) or cystamine and diisopropylcarbodiimide, respectively, in a mixture of dimethylformamide and diisopropylethylamine (∼10∶1) to generate a disulfide-linked dimer. The dimer was then reacted with either cystamine or N,N,N′,N′-tetramethylcystamine in the presence of a catalytic amount of cysteamine to give the final asymmetric disulfide-capped fragment, which was purified by HPLC. The identities and purities of all reaction products were verified by liquid chromatography (LC)/MS.
Plasmid Construction and Protein Preparation.
The catalytic domain of PDK1 (residues 51–359) was cloned into a pFastBacHTa vector and expressed in Sf21 insect cells. All mutants were generated by Quikchange mutagenesis (Stratagene) using standard protocols (see SI Text).
Disulfide-Trapping Conjugation Assays.
Immediately prior to disulfide-trapping experiments, PDK1 was exchanged into 25 mM Tris pH 7.5 using a Nap-5 size-exclusion column (GE Amersham) and fresh β-ME (sealed vials from Thermo Scientific) was added to the desired concentration (see Results). Proteins were incubated with disulfide compounds in 96-well plates at room temperature for 1 h prior to mass spectrometric analysis using a LCT-Premier LC/electrospray ionization-MS instrument (Waters). Protein masses were deconvoluted using the Max-Ent algorithm within the MassLynx software. Conjugation strengths were measured by comparing peak areas for the conjugated versus unconjugated protein.
Kinase Activity Assays.
A radioactivity-based kinase assay using as substrates [γ-32P]-ATP and a peptide derived from the activation loop of Akt (KTFAGTPEYLAPEVRR) was used to monitor effects of disulfide compounds on PDK1 activity (see SI Text).
Stoichiometric disulfide/PDK1 conjugates were prepared immediately prior to crystallization by incubating partially purified protein with an excess of disulfide compound in the presence of β-ME until conjugation was complete (as indicated by LC/MS). The conjugates were purified by size-exclusion chromatography and concentrated to 8.4, 8.4, or 16 mg/mL for the 2A2, JS30, and 1F8 complexes, respectively. To the JS30/T148C and 2A2/T148C complexes were added BIM-II at an approximate twofold molar excess. All protein complexes were crystallized by hanging drop vapor diffusion by mixing the protein with reservoir solution. Orange, hexagonal rod-shaped crystals of PDK1T148C/2A2/BIM-II and PDK1T148C/JS30/BIM-II were obtained at 20 °C using the following reservoir solutions: for 2A2, 0.1 M sodium citrate pH 5.4, 0.1 M potassium sodium tartrate, 1.8 M ammonium sulfate; and for JS30, 0.1 M sodium citrate pH 5.6, 0.2 M NDSB-211, 1.7 M ammonium sulfate. Square rod-shaped crystals of PDK1T148C/1F8 were obtained at 4 °C using the following reservoir solution: 0.1 M Bis-Tris pH 6.5, 0.2 M lithium sulfate, 25% (wt/vol) PEG 3350. Crystals were immersed briefly in cryoprotectant—for 2A2 and 1F8 complexes, a mixture of Paratone-N and paraffin oil, or for JS30, 0.1 M sodium citrate pH 5.6, 1.7 M ammonium sulfate, 0.2 M NDSB-211, 20% glycerol—and flash-frozen in liquid nitrogen. Diffraction data were collected at the Advanced Light Source, Berkeley, California (Beamline 8.3.1) and scaled with HKL2000. All structures were solved by molecular replacement (in CCP4) using, as a search model, the ATP-bound structure of PDK1 (Protein Data Bank ID 1H1W) (27). Structures were subsequently refined iteratively using WinCoot (28) and Phenix (29) (Table S2).
We acknowledge Adam Renslo and Punitha Vedantham in the Small Molecule Discovery Center at University of California, San Francisco (UCSF) for guiding preparation of the fragment disulfide library. We thank J.J. Miranda (UCSF) for instruction in protein expression. We thank Susan Taylor (University of California at San Diego) and Adrian Whitty (Boston University) for helpful comments and criticism. This work was supported by grants from the National Institutes of Health (R01AI070292 to J.A.W.), the Sandler Foundation (J.A.W.), and postdoctoral fellowship awards from the California Tobacco Related Disease Research Program (110385 to J.D.S.) and the National Institutes of Health (F32CA119641 to D.W.W.). The Advanced Light Source is supported by the director of the Office of Basic Energy Sciences, in the US Department of Energy under Contract DE-AC02-05CH11231.
- ↵1To whom correspondence should be addressed. E-mail: jim.wells{at}ucsf.edu.
Author contributions: J.D.S., D.W.W., C.L.M., M.P.J., and J.A.W. designed research; J.D.S. and C.L.M. performed research; J.D.S. and M.A.B. contributed new reagents/analytic tools; J.D.S., D.W.W., and C.L.M. analyzed data; and J.D.S. and J.A.W. wrote the paper.
The authors declare no conflict of interest.
Data deposition: Structural coordinates of PDK1 T148C bound to allosteric modulators have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3ORZ, 3OTU, and 3ORX).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102376108/-/DCSupplemental.
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Hytte mh20743
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Our Achievements
Header photo by Camille Munday
What we did in 2018
The Friends had another excellent year, raising money for a large conservation project to restore elm trees in the Park, funding four other conservation projects, continuing to grow its volunteer litter-picking group, helping in the adoption of Volunteer Community Rangers in the Park and developing a new website.
However, we continue to struggle to protect the Park against the rapidly increasing number of visitors who are encouraged by social and mainstream media. Deer harassment is widespread and indeed many people come just to see, and get close to, the deer.
We continue to educate people about how to treat the Park through our film and our Tread Lightly message, but it is an uphill battle. We hope that the new Volunteer Rangers will help to tackle the worst aspects of visitor behaviour.
Elm Walk
The Friends worked with The Royal Parks ("TRP") to create a new Elm Walk of 48 trees along the path leading from Petersham Gate. The project is intended to re-establish elms in Richmond Park using a hybrid resistant to Dutch Elm disease and provide a habitat for the White-letter Hairstreak butterfly. TRP designed the walk and managed the tree planting, while the Friends marketed them to potential sponsors and ran four "planting" days when donors were able to hold small ceremonies. We successfully found sponsors for all 47 trees and raised over £80,000 to fund the project, with the surplus going to other conservation projects in the Park. The first tree in the Walk is in the name of our patron Sir David Attenborough. The trustees are very grateful to all the sponsors and to the volunteers who organised the appeal.
Conservation Projects
The Friends funded four conservation projects in the Park during the year, often jointly with the Visitor Centre and sometimes with TRP also. They included:
- the permanent fencing of Ham Gate Pond which, after its restoration and the removal of invasive weeds, has become a haven for dragon/damselflies;
- fencing to protect the Pen Ponds shore line and its wildfowl from deer and dogs;
- fencing and planting in Pen Ponds Plantation to create a new deer-proof area along its land border where a native hedge has been planted, creating a new habitat for birds and insects; and
- permanent fencing around the gorse enclosures near Holly Lodge which have become a very successful bird habitat since they were created ten years ago.
Unfortunately, the second stage of the Beverley Brook restoration was delayed into 2019, to avoid harming the skylark areas.
Volunteer Community Rangers
Over the last three years the Friends has worked on the idea of Park Rangers to help deal with the impact on the Park of rapidly increasing visitor numbers, particularly as police numbers have declined so drastically; this included researching similar places such as the Peak District and Dartmoor National Parks. Discussion with TRP settled on a plan for Volunteer Community Rangers to educate the public and tackle inappropriate behaviour. TRP have now implemented that plan and recruited and trained volunteers, including some Friends' members, for a trial this Easter and a larger scheme in the summer. We think the Rangers could have an enormous impact on problems such deer harassment.
Visitor Centre
Some 60 volunteers staff and organise the Park's Visitor Centre (the "VC"), which provides information about the Park and items for sale, with the surplus funding Park conservation projects. The VC is open 364 days, from 10am to 4 pm in summer and 11 am to 3 pm in winter. In 2018 the VC had 55,000 visitors and £66,000 in sales. Park-related products developed by the Friends – calendar, Christmas and greeting cards, DVD, books and merchandise – contribute about half of the VC's surplus. We introduced collapsible re-usable coffee cups for sale to reduce plastic usage in the Park. Sadly, Mary Davies and Liz Hunt, who have been responsible for leading and developing the VC for the last nine years, stepped down at the end of the year: they will be sorely missed. The VC is a key to the Friends objective to educate the public about the special nature of the Park and to help visitors to appreciate its ecology, wildlife and heritage.
We now have a well-established and substantial team of over 50 litter-picking volunteers working in the Adopt-an-Area scheme. People adopt one of 29 areas, covering the whole of the Park, and pick litter at least once a week when they are walking there. In addition, there are roaming litter-pickers and some who concentrate on particular areas such as roadsides. Individuals, couples, friends, in one case a whole young family have adopted and identify with their area and there is a waiting list for areas! It's a tribute to the team and especially to Steve Sandham, who set up the scheme, that it is now being copied in other parks, including Wimbledon Common.
Practical conservation work
This team of 20-30 volunteers, does hands-on conservation and continues to work every Saturday in the autumn and winter months removing rhododendron ponticum in Pen Ponds Plantation. During the year, it completed clearing the deer accessible areas and, after the new deer-proof fencing was installed (see conservation projects above) helped to plant the new hedge along the border of the Plantation. It has also been involved in coppicing the gorse in the newly fenced gorse enclosures. A small sub-group of volunteers – the Beverley Brook Volunteer Group - has regularly cleared Beverley Brook of rubbish and been involved in various surveys, notably of ant hills and nocturnal small mammals. The practical conservation group always attracts new recruits eager to work in the open air and contribute to the Park and has now adopted the Green Gym name to reflect its physical work.
New website
The Friends now has a new website. The previous one (and our first website) was over twelve years old and, while it still looked good and many people liked it, it was difficult for us to operate and websites have moved on considerably both technically and in what people want from them. The new site has much greater use of high-quality photos and should be easier both for visitors and us; it also works much better on phones and tablets. There is a new facility for people to apply and pay for Friends membership online and to apply to be a volunteer. The Trustees are very grateful to Steve Sandham and his team for their hard work and to the web developer, Blue Flamingo in Twickenham.
We now have about 230 volunteer posts. This includes staffing the Visitor Centre, practical conservation work, the Discoverers education programme, litter-picking, walks and courses, Park monitoring and of course, the 25-30 volunteers involved in organising the Friends itself. Volunteer teams are largely self-managing and we keep the central direction "light touch" with minimum bureaucracy, while still meeting statutory requirements such as H&S and GDPR. The Trustees estimate that volunteers contribute 14,000 hours a year, worth nearly £150,000 based on the London Living Wage, to conservation and public education in the Park. Once again, the Trustees would like to thank the many volunteers who have contributed so much during the year.
The Friends regularly campaigns on issues affecting the Park, using a variety of approaches, from behind-the-scenes lobbying to public campaigns. As we mention above, our work over the last two to three years on Park Rangers bore fruit in 2018. Two Trustees, Vivienne Press and Nick Coleman, undertook research on signage, including an inventory of signs in the Park (we counted over 1200 of which 300 are environmental) and signage best practice in National Parks in the US, and elsewhere, and this has helped to prompt TRP's current signage review. Our successful public campaign in 2017/18 for increased protection of the view from King Henry's Mound to St Paul's had its first practical impact in the autumn when the Olympicopolis development at Stratford announced a reduction in the height of its two towers and more developments followed suit. In contrast we unsuccessfully objected to two residential developments just outside Roehampton Gate that will add significantly to light pollution in the Park.
Other areas
The Friends film "Richmond Park – National Nature Reserve", narrated by Sir David Attenborough, has now been viewed over 100,000 times on YouTube and another 400,000 times on the TV channel London Live which broadcast it three times over the 2018 Christmas period. We have also shown it at thirty schools in the surrounding three boroughs.
We now have three sponsors - Russell-Cooke, Thames Water and The Property Partnership. They have their logo on all our communications, as well as other FRP publicity about what they do. Sponsorship provides us with a steady annual income of £7,500 that means we can plan ahead for funding conservation projects. We are very grateful to the three organisations for their support.
We offered over 20 public walks and courses (a talk and a walk) for adults, i.e. nearly two a month, with large numbers turning out for the Boxing Day walk and some courses. We are extending this to include more talks without the walk and tours of areas of the Park. We will keep the public walks free as a key part of achieving the Friends charitable objectives.
Discoverers, our education programme for young families, held activities such as Bat Watch and Fungi Art Workshop, but had to postpone some events because of poor weather. Two of the Discoverers families became involved as Butterfly Surveyors and we would like to encourage young families to become more involved with the Park in this way.
GDPR - We made changes to our data protection to meet the new GDPR rules. The rules seem to have been designed for large organisations and there was no relevant practical government guidance. It caused us (and other small charities) a lot of work for little benefit since we already had tight data protection. Our basic principles remain that we never share our membership and volunteer data with outside bodies, we have tight internal rules on who can see what data and we quickly unsubscribe people if they ask us.
The Friends' membership showed a small (3%) increase to 1780 memberships or 2600 members (counting one member for an individual subscription and two for a household subscription). Our turnover of members is very low at about 8%, implying our members stay with us for an average of 12 years.
Plans for the future
The Friends plans to maintain its activities broadly along the present lines. Our priorities in 2019 include developing a three-year plan for our funding of conservation projects, bringing in new people to help run the Friends, finalising the improvements in our volunteering process and helping TRP where necessary to implement the Rangers and signage improvements. We will also campaign strongly to protect Richmond Park against threats to its peace, natural beauty and wildlife, of which the latest in 2019 is likely to be Heathrow and its flights paths.
What we did in 2017
The Friends had an excellent year with the completion and launch of its film about Richmond Park with Sir David Attenborough, the development of a substantial volunteer litter-picking group and a big increase in donations and funds available for conservation projects. However, we struggle to protect the Park against the rapidly increasing number of visitors; the worst aspect this year was large-scale deer harassment by the public. The film, with its Tread Lightly message is our attempt to educate people about how to treat the Park but we are fighting a wave of mainstream and social media encouraging people to visit it. We want visitors to come but not at the expense of the Park's fabric and wildlife.
The Friends film "Richmond Park – National Nature Reserve" was launched in April at the Royal Geographical Society. Narrated by Sir David Attenborough and filmed by George Chan and Gavin Thurston, both of whom have worked with Sir David on his wildlife productions such as Planet Earth, its aim is to change people's perception of the Park from seeing it as just a local park to valuing it as a unique place with a rich ecology and wildlife that should be protected. Over 600 people, including 250 schoolchildren, attended the launch, which was managed by 50 volunteers and hosted by Sir Trevor McDonald. It featured Sir David himself, Lloyd Grossman the Chair of The Royal Parks (TRP) and Julian Glover the actor, who read David Harsent's lovely poem about the Park, commissioned by the Friends. The 20 minute film and the three minute social media version has now been seen by nearly 200,000 people in the six screenings by the TV channel London Live and online. A DVD of the film and the launch event is on sale at the Visitor Centre and we have offered a free copy to all schools in the three boroughs surrounding the Park. We continue to promote it as the centrepiece of our Tread Lightly campaign.
Begun by three members, including one of the Trustees, the Friends new volunteer litter-picking has quickly grown into a substantial activity and contribution to protecting the Park. It started with a Spring Clean of the Park in March undertaken by 85 volunteers to coincide with Keep Britain Tidy's annual event. Because many volunteers wanted more regular litter-picking, we set up Adopt-an-Area, whereby people adopt one of 29 areas covering the whole of the Park and pick litter at least once a week when they are walking there. Individuals, couples, friends, in one case a whole young family have adopted and identify with their area; there is now a waiting list for areas! We thank everyone involved for its great success.
Finances and conservation projects
Visitor Centre
Campaigning and monitoring
Other areas
The Friends' conservation volunteers have nearly finished their work removing rhododendron and planting trees in Pond Plantation; the Friends is funding fencing to protect replacement planting from deer. Our Discoverers programme for young families is in its fifth year and had events such as Park in the Dark sky-watching, a deer "safari", a bat watch, tree identification, fungi art and an Isabella trail.
We also offered over 20 public walks and courses for adults, i.e. nearly two a month, with large numbers turning out for the Boxing Day walk and some courses. The weekly Friday informal bird-watching walks, which were started in 2014, continue to be very popular. The Trustees intend to keep the public walks free and see them as a key part of achieving the Friends' charitable objectives.
The Friends' calendars, Christmas and other cards continue to sell well through the Visitor Centre and merchandise (tea towels, caps, hats, bags) which we started in 2016 has sold reasonably well. Surplus funds go to conservation projects in the Park.
Russell-Cooke, the local law firm, renewed their sponsorship for a further two years and we have added Thames Water as a new sponsor, with a third sponsor likely in 2018. Each sponsor pays £2,500 a year to have their logo on our communications and some other benefits. Sponsorship provides us with a steady annual income that means we can plan ahead for conservation projects without relying on income from donations, which can vary substantially from year to year, or on specific appeals
Membership - after two years of decrease following the change in subscription fee in January 2015, the Friends' membership showed a 7% increase to 1720 memberships or 2525 members (counting one member for an individual subscription and two for a household subscription) by end 2017 – higher than before the subscription increase.
Plans for the future
What we did in 2016
Visitor Centre
Practical conservation
Friends publishing and merchandise
Walks and courses
Plans for the future


text:
by skycloudnz
poem from life
|Growing older has many sides,
The family grows, the family dies,
The body grows weaker,
The mind is the speaker,
Where we cannot do,
We can help others do.
They say things have changed,
From when we were young,
Technology yes but people still dumb.
Like Socrates said many years ago,
The youth of today are wild and slow.
Progress is still hampered by personal greed,
The world should be better for those in need.
An economy based on dollars alone,
Needs people put into the woeful tone.
All our systems are so past their time,
We cling to them when change is the wine,
Needed to see the future together,
Left and right are old birds of a feather,
That put us to fight each other,
Get on together and fight for each other.
Pollution is not just physical.
The mind is needing musical,
Muse not just amuse.
It is people that have forgotten,
The wisdom of the old that have rotten.
Don't count on politics do it yourself,
People together can tidy the shelf,
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Use knowledge and technology,
Then the rhyme will come.


text:
It's not the first all-female crew in the history of the race, but this is the first time an all-female team will receive the same support as their male competition - from start to finish.
Why an all-female team? Globally, about 80 percent of the retail products SCA makes are purchased by women. This gives SCA a natural interest in the role of women in the hygiene of millions of families around the world. By entering this team in the Volvo Ocean Race, SCA is highlighting the strength of women in society and the ability of women to take on new challenges -- be they at home or at sea.
With nine months to go to the start, Team SCA is in full training mode - both physical and sailing - at their training camp in Lanzarote, Spain.
Émission animée tous les jours en direct à 18h par le journaliste-navigateur Pierre-Yves Lautrou
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text:
NARGES BAJOGHLI is Assistant Professor of Middle East Studies at the School of Advanced International Studies, Johns Hopkins University, and the author of Iran Reframed: Anxieties of Power in the Islamic Republic, forthcoming from Stanford University Press.
It was late in the afternoon on a cold winter day. Light snow had covered Tehran the night before, and I was spending the day in the production office of a small film unit of the Basij paramilitary militia. I was researching cultural producers in the Islamic Republic's [1] military and paramilitary organizations, and the young men who worked in this film unit had agreed to talk to me on the condition of anonymity.
Ali, a 20-year-old Basij film editor, came into the office where I sat with his colleague Mustafa, 24. Ali was working on a sequence for state television about U.S. media and its opposition to the Islamic Republic. Carrying his laptop, he excitedly showed us his latest find, a segment on CNN prior to the signing of the Iran deal about why the Islamic Revolutionary Guard Corps (IRGC) [2]and Basij are so powerful in Iran. The segment depicted Iran's armed forces as a homogenous crowd of bearded men with stern faces, dressed in fatigues or black button-down shirts, listening to the supreme leader. Mustafa and Ali watched the report with wide smiles. Ali turned to me once it ended and said: "It's funny to me how Westerners depict us. They make it seem as if the leader [Ali Khamenei] says something and we just fall in line." He laughed. "Does anything in Iran work so smoothly? They should come and see how messy everything is here. What makes them think that amid everything barely functioning––from our economy, to our traffic, to our work culture––the Basij are so well organized?"
Across town the following week, I observed a production meeting with a group of IRGC leaders in charge of media. All the men in the room were in their early 50s. Fervent revolutionaries in the first decade of the Islamic Republic, today they wanted to modify the system by easing social and cultural restrictions and opening the political system to greater competition, but they disagreed
[1] https://www.foreignaffairs.com/articles/iran/2019-01-10/why-iran-waits
[2] https://www.foreignaffairs.com/articles/iran/2017-06-13/how-deep-irans-state


text:
Symonette: This is the Christian thing to do
Minister of Immigration Brent Symonette said yesterday that it is The Bahamas' Christian duty to assist Dominica in its time of need.
Prime Minister Dr. Hubert Minnis revealed yesterday that The Bahamas will accept students and other Dominicans with families in The Bahamas as that country begins to rebuild after the devastation of Hurricane Maria.
The Nassau Guardian reached out to Symonette, who is coordinating the exchange effort with CARICOM, and asked whether the Minnis administration is concerned about any backlash from the public.
"We are being Christian to our Caribbean neighbor, who was devastated in a major hurricane," he said.
"When you listen to the prime minister speak, Prime Minister Roosevelt Skerrit, and just look at him – because I've known him for several years – he really looks like he had the whole world on his shoulders.
"It could be us one day, too. For us to extend a loving, friendly hand to a Caribbean sister nation, if people have a difficulty with that, we cannot justly call ourselves a Christian nation."
Symonette said he will be talking through CARICOM on how the exchange will work.
"You raised the question of if we are xenophobic. The way forward for The Bahamas in the world scene is to get over our hang-ups in that area, if we are ever going to develop as a country," Symonette said.
"Until we are prepared to accept that there is a world beyond The Bahamas, we will never develop as a first-class country.
"We had our devastation on Ragged Island, but in Dominica it's a whole country.
"We have to accept the magnitude of it.
"The schools are destroyed; the hospitals are destroyed; the prime minister's roof came off.
[Members of Parliament] are coming to work by fishing boat."
Skerrit, who landed on New Providence shortly after 10 a.m. yesterday, briefly met with Minnis and Symonette at Jet Aviation. He was in transit to Dominica.
On Saturday, Skerrit delivered a speech before the United Nations General Assembly on climate change and the state of his country.
"I want to thank the government of The Bahamas for offering to host and facilitate the schoolchildren, especially students, to be able to come here and to be housed by families," he told The Nassau Guardian.
"This will certainly help a number of families in my country."
Travis Cartwright-Carroll
Education: College of The Bahamas, English
Latest posts by Travis Cartwright-Carroll (see all)
- Fueled by arrogance - December 18, 2019
- Davis gives notice on no confidence motion in PM - December 5, 2019
- Where do they go? - October 16, 2019


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Boys Sophomore Baseball · Sophomore Baseball Change
The sophomore baseball game today vs. BC has been changed to Burlington at 4:30 PM.
The sophomore baseball game today vs. BC has been changed to Burlington at 4:30 PM.
The sophomore baseball game today (4/4/19) vs. Belvidere North has been moved to Belvidere North. Start time is 4:30 PM.
The start of the Sophomore baseball game @ Hampshire today (3/27/19) has been bumped back to noon. The bus will now leave at 10 AM.
Click on the link below to access the Spring Parent/Player presentation that was held on 2/21/19. https://docs.google.com/presentation/d/1KDT7DtosOtniMGFFaDR4ZIbRllJlBli5ctPnzy8C2CE/edit#slide=id.p
Sports team picture day is coming up on 3/6/19! If your son or daughter didn't bring home a picture form for you to fill out, you can still order pictures. The athletic office has extra forms. Or you can pay ahead of time online by going to https://www.vipis.com/group/cb463f60/woodstock-high-school If you pay online, please let your athlete know so they know to have their picture taken. If you have any questions about sports team and individual pictures, please
It's time to get your fall athletes registered. Fall sports are now open for registration online. Go to www.bluestreakathletics.com Click the MORE Tab Click REGISTER AN ATHLETE Click on REGISTRATION -First-time users, create an account. This will stay with you forever. -Return visitors, login Click on Begin Registration, and follow the prompts


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(New Delhi)Saffrons & Broomers are Boomeranging Air Pollution in Delhi
Delhi's air pollution skyrocketed after Diwali. A layer of haze enveloped the national capital as the city's air quality plummeted to the "severe" category for the first time this season.
The overall air quality index (AQI) stood at 463 at 11.30 am, according to the System of Air Quality and Weather Forecasting and Research (SAFAR).
The AQI at Pusa, Lodhi Road, Airport Terminal T3, Noida, Mathura Road, Ayanagar, IIT Delhi, Dhirpur, and Chandni Chowk was 480, 436, 460, 668, 413, 477, 483, 553 and 466, respectively. Ruling AAP on Monday said that the saffron party has "a habit of" opposing everything that the Kejriwal government does.
But Delhi BJP leader Vijender Gupta hit back, saying the Aam Aadmi Party is making these claims to "distract" people from its failures to curb air pollution to a manageable level.
Addressing a press conference, senior AAP leader and Rajya Sabha MP Sanjay Singh said BJP leaders should think about their families before bursting crackers.
He was apparently referring to Delhi unit BJP chief Manoj Tiwari, who posted a video on Twitter where he could be seen lighting earthen lamps and bursting firecrackers with people in unauthorised colonies.
Gupta said ,AAP is making such claims because its government has not done any ground work in Delhi. "Controlling pollution needs work all through the year. The AAP has not done that and now to distract people from its own failures it is making such comments."
Tag: AAP's Delhi Govt
Indisposed CM Kejriwal Approves Pay+Allowances Hike of Delhiites 70 MLAs
[New Delhi] Indisposed CM Kejriwal Approves Pay+Allowances Hike of Delhiites MLAs
This decision was taken at a Cabinet meeting chaired by Indisposed Chief Minister Arvind Kejriwal at his residence.
They, however, did not specify the quantum of hike in allowances and salary. Delhi has 70 MLAs.
In July, a group of AAP legislators had demanded a significant hike in salary, arguing that their earnings were not enough to run their family and offices.
In October, a three-member committee headed by former Secretary General of Lok Sabha PDT Achary had recommended two-and-half-fold hike in salary from current Rs 88,000 per month to Rs 2.10 lakh.
The committee, which had submitted its report to Assembly Speaker Ram Niwas Goel, had favoured a 400 per cent hike in basic salary of MLAs from Rs 12,000 to Rs 50,000 per month.
AAP's Delhi Govt Syphoning Fatty Cakes to Its Party Members
[New Delhi]AAP's Delhi Govt Syphoning Fatty Cakes to Its Party Members
Aam Aadmi Party (AAP) government in Delhi today admitted that a few party members were getting benefits like official house and cars.
In a reply to BJP MLA OP Sharma's question, government said that these members have been appointed as co-terminus staff with it.
Government in its reply said that Bibhav Kumar, private secretary to Chief Minister Arvind Kejriwal and Aswathi Muralidharan, joint secretary to Chief minister, have been allotted houses.
The government also said the salary of these staff, who are in the grade pay of Rs 8,700 equivalent to senior IAS officers.
Apart from 17 co-terminus staff, eight contractual staffs are hired and get Rs 60,000 to Rs 1.15 lakh.
There are two advisors who work on the salary of Rs one per month.


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A pair of photos from the National Guardsman magazines of 1956 with one Captain Norma Parsons. Parsons, a CBI vet, was apparently the first woman officer in the Air National Guard from any state.
And this online period find of her, haven't found an OBIT of her, even using her apparent married name, Erb, could she still be around!!


text:
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Womack opens his as-told-to-memoir stripped down to his skivvies and hunted by his wife with a gun. He plays dead after the gun goes off, and then runs when his wife leaves the scene. "I bumped into cops scoping the neighbourhood [sic] for housebreakers and other likely fuck-ups."
Two pages after that, a Cleveland childhood "so ghetto" that the rats "walked past and said, 'How you doin' man?'" sets the scene. Turn the pages, though, and a weirdness seeps in—no payoff, no slam-bang in-the-spotlight "I made it" moment.
Womack grew up broke, got into singing but still couldn't get laid, married the widow of his best friend Sam Cooke (she's the one with the gun), slept with his stepdaughter (who went off with his brother Cecil and became a star alongside him), barely dodged bullets, stuck his nose into nose candy, said goodbye to Janis Joplin right before she said hello to the dealer with her fatal dose, hung out with the Rolling Stones (but they wouldn't let him use the limo)—and through all of it, no payoff, no pleasure—only hard times.
Informative, even with all the British-isms thrown in—but frustrating. Nowhere can you intuit the man who grabbed "Sweet Caroline," stripped it down to its gospel roots with his own gospel roots, pumped it full of climaxes, and ran with elation towards some bright light of ecstasy.
As his 2014 Jazz Fest set demonstrated, he deserves his legacy. But you need "Sweet Caroline" and the other records to know that.


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The Forty-Nineth Annual Grand Master Fiddler Championship will be held on Saturday September 5th and Sunday September 6th, 2020 (Labor Day Weekend), in Nashville, Tennessee, at the Country Music Hall of Fame and Museum in the Ford Theater. Contestants from all over the world will come to compete. In 2019, Ridge Roberts of Texas claimed the Championship. Who will the 2020 Champion be?? Please plan to join us for the competition!
On Saturday, the GMFC welcomed our special guest Ketch Secor, the fiddle player and founding member of the Grammy award winning group Old Crow Medicine Show. Ketch will be serving as a judge for the Traditional category and will be performing in the afternoon.
On Sunday, the GMFC welcomed back our youngest champion ever, Mark O'Connor. Mark was honored with the Dr. Perry F. Harris Distinguished Fiddler, for his contributions and support for fiddle and fiddle music. Mark and his wife Maggie did an amazing performance on Sunday afternoon, and had a lot of great information about the O'Connor Method, a new American School of String Playing.
The Grand Master Fiddler Championship Inc. is organized as a Tennessee nonprofit and U.S. IRS 501(c)(3) charitable corporation, formed to educate about and perpetuate fiddling as an art form and cultural treasure. The corporation's primary focus is holding the annual Grand Master Fiddler Championship competition - an event that has started many young musicians on their way to a career in music. As a 501(c)(3) entity, all donations are tax deductible, and we welcome your support to promote and educate about fiddling. Fund-raising efforts are being planned in order to expand the competition, enhance our educational efforts, and to further the awareness of fiddling around the globe.
Fiddling laid the foundation of the WSM Grand Ole Opry and remains important to country and bluegrass music today. For a quarter-century, the contest was sponsored by WSM, the Grand Ole Opry, the Country Music Association, and later by Gaylord Entertainment. When financially able, the GMFC makes donations to such charities as the Grand Ole Opry Trust Fund and the Bluegrass Trust Fund which benefit musicians in need. We do this in honor of the contributions of the pioneers to country, bluegrass and traditional music. Many of these pioneers such as Roy Acuff, Porter Wagoner, "Uncle Jimmy" Thompson, Bill Monroe, Howdy Forrester and Tommy Jackson played the fiddle or were very involved with fiddling. We have also donate fiddles/violins to the W. O. Smith Community Music School in Nashville.
Please tell all your fiddle music friends, websites, discussion lists and other music sites about this web page! Let us hear from you and we will add you to the GMFC email list.
Help Support the Arts in Tennessee by participating in the Tennessee Specialty License Plate Program benefitting the Tennessee Arts Program - proceeeds from every license plate go to help fund Arts in Tennessee. To get more information or order your plate please see http://tn.gov/revenue/article/tennessee-arts-commission.


text:
Summer may be a time once folks feel a lot of relaxed, laid back, and within the mood for fun. Summer maternity garments follow this a lot of relaxed, casual, fun trend. Summer maternity clothes need to perform many functions. First, they need to keep the wearer cool and comfortable. It's already bad enough being big and pregnant throughout a hot summer so you don't need to deal with uncomfortable, hot, constricting clothing. Second, summer maternity clothes need to work for a wide variety of purposes from work to summer barbeques, and poolside parties to the beach. Finally, summer maternity clothes need to make the wearer look and feel fantastic. Fortunately, the latest summer maternity trends can serve all of these purposes!
Maternity dresses with lots of color are cute, comfy, and trendy for summer. Vibrant maxi dresses paired with sandals square measure a must have explore for summer. Shorter, knee-length dresses look updated with ruffled hems, floral prints, and tie-dye. All of these looks are very versatile and can be dressed up or down with the right shoes and accessories.
Tanks square measure fantastic summer maternity wear as a result of they appear cute, they can be dressed up or down, and they keep you nice and cool on hot summer days. Printed tanks are often paired with shorts and sandals for a grounds barbeque or dressed up with a maxi skirt for a night out. Ruffled tanks will look fancy with a good combine of linen pants, and you'll be able to forever throw a bit cardigan over your tanks to feature interest and to stay you warm on breezy summer nights.