text:
This has been one long Summer, and now I can relax a bit and enjoy the Fall and it's cooler weather--not that our weather has been particularly hot this year.
I spent a good part of my time getting ready for two shows that I was looking forward to doing and was working almost every weekend to make more stuff for the shows. However, fate had other plans.
About a week before my first show in August, I started to feel a slight twinge of pain in my stomach, but only when I moved in certain ways. Not really thinking much of it, I continued about my business as usual. On the Monday before my show, I went to work as normal and after work went for a bike ride through the wooded trails near our house. For two hours and rode all around the woods and enjoyed the summer evening.
After my ride, I came home and ate dinner--all the while feeling a bit uncomfortable but not really paying much attention. During that night, however, the pain increased and I had some trouble sleeping through the night.
In the morning, I got up, packed up my lunch and breakfast, got ready for work and just before leaving, told my partner that I was having some minor pain in my stomach. He suggested calling the doctor and I agreed. I promised to call the doctor and set up an appointment when I got to work.
Once at work, I started getting an upset stomach and couldn't bring my self to eat my breakfast. Then I knew I was sick. I was turning down food.
I called the doctor and left the symptoms with the nurse and she promised to call me back within the hour. Ten minutes later I was called back and told to go to the emergency room.
I figured they just couldn't fit me into their schedule, so I still wasn't thinking anything big was going on. I told my boss about the issue and headed back home so I could change into comfortable clothes and pick up my partner before going to the hospital. Still figuring I would be back at work the next day.
In the ER, they were having some trouble diagnosing my issue since it wasn't on the right side and I wasn't having nearly enough trouble for a normal appendicitis. However, the CAT scan revealed that it was indeed my appendix and it had to be removed--immediately.
"Will I be able to do the art fair this weekend?", I asked.
"I wouldn't recommend it," was their prompt reply.
So, after paying $500 for my booth at the show, I could not attend. Not only did I lose the money, but I'd spent a long time working on items that I just couldn't take to any show.
To say I was depressed would be an understatement, but I focused on getting better and was soon back on the torch to get ready for the next show.
Another month of torching, and I was ready to display a wide range of heart and leaf pendants along with some of my older items too.
The show ran three days over Labor Day weekend and it was my first time there. While first couple days started off slow, sales were steady until the last day.
I began to panic a bit, but I needn't have. As the afternoon wore on, I ended up having my best day of the show and doubling the sales for the weekend.
It turned out alright, but I'm still upset that I missed one of my shows and lost all of that money. Hopefully, next year's shows will work out better and I can avoid emergency surgery.
So, what's your art fair horror story?


text:
Dr. Akpan is an Associate Professor of Secondary Education (Science). Dr. Akpan holds a Ph.D. from Iowa State University in curriculum and instruction with cognates in science education. His work at JSU has included teaching undergraduate and graduate science methods courses, practicum observations, and internship reflective practices. Dr. Akpan advises graduate and undergraduate students in science education programs. His approach to the learning and teaching of science is based on a constructivist approach, which both challenges and engages the learner. While he sees science as being very much a hands-on activity, he likes to take this a step further by making science a minds-on and knees-on activity. Teaching, researching, advising, service and preparing teachers for the classroom are Dr. Akpan's passion.
Dr. Akpan's current research interest focuses on investigation of how technologies can be used to improve teaching and learning of science, as well as the nature of science.
Dr. Akpan's current teaching assignments include Critical Inquiry in Science Education, General Science Education, Education Methods, Assessment and Technology in Secondary Schools, Psychological Principles of Learning, Practicum Observations and Internship Reflective Practices, Foundations of Education, Philosophy of Education and Foundations of Secondary Education.
Jacksonville State University
Secondary Education
Suite A-6 Ramona Wood

subset: commoncrawl


text:
The automotive equivalent of a lost Picasso - a 1954 Formula 1 car driven to victory by Juan Manuel Fangio - has been found buried at the back of a shed.
Believed lost for over three decades, the 1954 Mercedes-Benz W196 will be up for auction at this year's Goodwood Festival of Speed in July.
It's expected to fetch more than $7.3 million.
Fangio piloted this silver arrow, designated chassis 00006/54, to victory at the Swiss Grand Prix by over a minute.
It's also a winner at the fearsome Nurburgring.
Powered by a naturally aspirated 2.5-litre straight-eight, the W196 was the car to introduce fuel injection, fully-independent suspension and tubular-space frame construction into Formula 1.
Mercedes-Benz is tipped to be a leading contender to purchase the car, with the brand reportedly keen to add it to its already extensive collection of rare racers.


text: On November 9, 2003, HAROLD F. GRAUL SR., husband of Nancy T. Graul. Funeral on Thursday, November 13 at 11 A.M. in the John M. Taylor Funeral Home, 147 Gloucester Street, Annapolis, MD. Interment Hillcrest Cemetery. Family will receive friends on Thursday, November 13, from 9:30 A.M. until funeral time at 11 A.M. in the funeral home. Contributions may be made to the Hopice of the Chesapeake, 8424 Veterans Highway, Millersville, MD 21108 or to the Geaton and JoAnn DeCesaris Institute, 2001 Medical Parkway, Sajah Building, Annapolis, MD 21401.


text:
Update: Charles Wang did the right thing in taking action with Chris Simon before the league, and by being the mouthpiece for the organization. As a heavily involved owner, it was only right for Wang himself to address the status of Simon rather than drop the obligation in the lap of Garth Snow, or worse, Ted Nolan. Snow and Nolan will undoubtedly have to field questions on the matter, but they will do so with the official position of the organization clearly defined.
As for the content of the statement, I'm sure it will anger many NHL fans, as well as Islander fans, that the team didn't immediately sever its ties with Simon. Contract and CBA issues aside, it's apparent that the organization views Simon not as a threat to its integrity but as a troubled member of the family. It is not a sin to be loyal and supportive of someone in need. However, if Simon's acts of unbridled aggression on the ice are symptoms of problems that require counseling, then he should enter counseling with the goal of preparing himself to live a productive life beyond that of a hockey player. That life may be right around the corner, pending the league's involvement.
The Islanders should not saddle themselves with expectations of when Simon will be ready to return and how he will fit back into the team's picture. You can only apologize and say "there's no excuse" so many times. The team needs to move on and address its on-ice goals without such distractions. There are jobs to be won and roles to be established. From this moment on, the Islanders and Chris Simon are fighting very different battles.
The severity of Chris Simon's punishment depends on neither the force of the blow nor the extent of the injury it caused. Simon has demonstrated once again that he has no regard for the well being of another human, even if that human is a hockey player of questionable ilk like Jarko Ruutu. Whereas another player would have dismissed Ruutu with a shove, a face wash, or even an invitation to engage in legitimate combat, Simon chose to expose his adversary's flesh to the forceful delivery of a sharp blade.
It does not matter that the target of the blow was protected by a sturdy skate boot. The act was heinously irresponsible and within in an inch or two of causing a catastrophic injury. The NHL cannot tolerate such a degree of callousness.
Maybe in Simon's mind there is a difference between Ruutu and other players. Maybe Simon believes that players like Ruutu and Ryan Hollweg are so low that brutalization and mutilation are somehow justified. If this were an isolated incident, we would be searching for an explanation. What did Ruutu do to incite Simon's wrath? Wasn't Simon just being a good teammate, protecting his boys? But the act is indefensible and so is the body of work.
Following the game, one could sense the hesitancy to react among the players in the locker room. Certainly, they were unclear about exactly what had transpired. Additionally, they appeared to disbelieve that this had happened again—a selfish act that compromised their ability to win a game, and would put them in the awkward position of having to explain the detrimental actions of a teammate. It's tough to put your teammates through that once. To make them do it twice in less than a year leaves little room for recovery. How fast will they be lining up for the next round of apologies?It's almost painful to think about how Ted Nolan must feel in this situation. He must know that no one wants to hear an unqualified statement of support for Simon. But given their well-documented personal relationship, one has to expect that Nolan will express some sort of concern for his once-troubled protege. It is sure to be a delicate balancing act. You wonder where the middle ground is among understanding, imposition, and a sense of betrayal.
Time permitting, I'll have some notes from the game itself later today. Islander Frontier has an excellent piece on the Simon story that is well worth reading regardless of your perspective.


text:
This set is a commissioned pieces for a client. She wanted a simple set with pearl.. something simple with no filigrees but with lots of details.
Then I decided to try all of Gailavira's tutorial that I bought sometime ago but did not get a chance to try it.
So here they are..... GAILAVIRA'S CARNIVAL PENDANT
This ear studs is a modification of GAILAVIRA'S ETERNAL SANCTUARY.
And GAILAVIRA'S SENSU RING.....
Look at them ...... they really looked adorable..... =)
I can assure you that my client is one happy customer..... thanks to Gailavira ....


text:
Accountrak was designed to meet the needs of a not-for-profit organization. The program was developed with the help of CPA's to provide you a fund accounting solution that will meet and exceed your expectations.
Reviews of Accountrak Write a Review
No reviews have been submitted. Do you use Accountrak? Write a review, and help other organizations like yours make smarter, more informed software selection decisions!


text:
I couldn't find any blogs about the weekend program, so I thought I might share our experience.
So after little steps at Monash Council, we went on an overseas holiday (major disruption to routine), had different caregivers (who prefer that babies don't cry), I went back to work... So by 10months, baby was rocked to sleep for her day naps, sleeps on a person for the second cycle, rocked to sleep at night, and needed more rocking every time she woke up, and after 12am, I succumb to giving her the breast to suck while I keep sleeping beside her. We were needing to get up about 4-6times overnight.
So the decision to go to Masada to get some help. I didn't want to take a week off work, so opted to do the 2weekend program. The aim was to get someone to break the sleep associations that the baby had.
We got admitted Saturday afternoon, talked about our goals and routine and how to adjust the routine to an age appropriate routine. We handed baby over to help with the afternoon sleep, then the nighttime sleep. I was fortunate to have a room away from the babies, so I couldn't hear them cry (which is also odd for me). There were only 8 of us so it wasn't a busy and noisy ward.
The next day was us settling the babies and the staff teaching us what to do. I must say, I didn't get to settle the baby at all because she didn't hit the time for a grizzle.
Discharge was on Monday at 0800. I had intended to go to work but decided that bad city traffic and wanting to implement the routine at home, so I took Monday off work. I should have planned it better and taken that day off anyway.
We stuck to the routine and principles and managed a week with good day and night sleeps. I was wondering if I needed to go back to that second weekend and decided that we needed to consolidate what we had learnt.
Baby performed amazingly and sailed through the second weekend.
Now is the third weekend and we are doing 7.30pm to 6.30am night sleeps with no rocking, no breastfeeding, no patting. She is relieved to be in the cot. Hopefully she keeps going well!


text:
I have white carpet – so you know I did not embark on this 'bare-bottom, early potty training' thing lightly. I researched the different options for potty learning for toddlers and this seemed the right one for us. Yes, it is intensive but that kind of full-on, go for broke approach works for me. I am very much that type of person: jump in with two feet, go all out and then take a break and focus all that energy on something else. Yep. "I can totally do three very intense, non-stop, bottom-watching days if that means I am pretty much done after that", I thought. Good thought, right?
It has been three weeks since we started this '3-day' potty learning method (apparently I am not allowed to use the words' 3-day potty training' – see comments for details). I want to let you in to how it went for us. I want to write the post I was looking for before we started this method – a 'warts-and-all, how it really happens' post – a post I could not find at the time. If you remember, this is a potty learning approach that promises that in three days your little one will get the hang of it and that in ten days to two weeks there will be few or no accidents. It is based on working with your toddler's natural desire to be naked/diaper-free and is a gentle approach that does not use threats, shame or tangible rewards to bribe your child (more on that later) and it is said to work best on kids between 15 and 27 months of age.
Here is a post in which I outline how this method works and why we felt it was for us. Nica (uh, still working on that nickname) was 19 months old when we started our 3-day intensive potty-learning kick-off weekend.
I have heard online from a few friends for whom this approach worked perfectly. This is the reality of where we are at, now:
- We have been a diaper-free household for three weeks (technically we have been 'day-diaper'-free for three weeks and have been completely diaper-free for over two weeks, now.)
- Nica seems to be fully night trained and actually now sleeps bare-butt (accident-free). I was not expecting this. It just happened. On day two (!!!) her morning diaper which previously was always really, very full was completely empty. I thought it was a fluke and kept putting night-diapers on her for the first few days until I realised they kept being dry, so I decided to take the leap. We have been completely diaper-free, including for naps and night sleep (with no wake-ups for potty) since then.
- She is 60 to 95% home-trained – meaning that if we stay home she gets between 6 and 9.5 out of every 10 pees in the potty (yes you can get only half a pee in the potty… trust me). It varies widely from day to day, though. The main 'misses' happen when she is stressed, tired or distracted – and to be fair there has been a lot going on, the last few weeks.
- We have had very few poop-misplacement incidents (PMIs – hah). We have, over the last three weeks, had one or two but it is clearly much easier for her to recognise that a poo is coming and to want to get it in the potty.
- We can safely go out for short periods of time of up to an hour or two, wearing nothing but loose fitting trousers (no pull-ups, underpants or diapers), reasonably confident that there will be no accidents.
- We can go out for longer periods of time, taking a potty with us and Nica will either tell/show us she needs to go (by tugging at her trousers and saying 'off, off' or by saying 'potty' or 'pee-pee') or she will go when I offer her the potty.
- … However, we are yet to have a completely 'spill'-free day (though we have come very close, a few times).
- She can hold it in overnight
- During the day, Nica can sense that the urge is coming and spontaneously walk to the potty and do her business and/or…
- She can communicate when she needs to go with a few seconds lead-in (30 seconds at most, I would say, still) so I can pull her trousers down and get her to the potty
- If she does start peeing somewhere other than the potty and NinjaDad or I spot it, she will stop midstream and hold it in until we get her over the potty
- She can pee when prompted (i.e. if I ask her to sit on the potty, she will and if there is a need, she will pee then)
… she can do all of those things but that doesn't mean she always does… And so it goes. There are still good days and bad days. Today has been a great day. For most of her pees and for her daily poo she walked to the potty herself (spontaneously and unprompted) and did her thing and then often helped me go and flush it away, too. She also went, when prompted, in the potty while we were out (once in the boot of the car, once in a public toilet at a shopping center). She did have two small accidents: the first was when she was down for her nap, lying down but not asleep – it seems to be much harder for her to anticipate that a pee is coming when she is sitting or lying down; the other was when we just got home after going to the store – she said she needed the potty, I just couldn't get her there in time… So, especially that last accident, actually doesn't discourage me very much at all: after all she still knew it was coming and communicated it to me, even if I just couldn't get her trousers off and move her to the potty quite quickly enough. We have been having more and more of these terrifically successful days – they are not 100% accident-free but I am still so proud of her EVERY time she goes in the potty, spontaneously of her own accord. It is just great.
There have also been days when she still demonstrates many of these skills (like walking to the potty when the urge comes, unprompted) but mixed in there are some spectacular cases of her acting like she has never seen – or doesn't want to see – a potty. Yeah, just as soon as I thought she was taking it all in her stride, she suddenly would refuse the potty when offered and then pee on the floor two minutes later or something like that. More distressing still, a few nights back she started yelling 'no potty, no potty, no potty' in her sleep… anxiety-dream?? That made me so sad. I'd like to think she was just processing this strange, new change in her daily habits. It is a big change. I mean, she also likes to take her bear to the potty – where he can poo, fart, pee (complete with sound effects) – and then she walks him off to the toilet to flush the pee away, wipe his bum and wash his hands. Adorable – and clearly another way of processing the shift in behaviour! But her night shenanigans made me wonder if she was more stressed about it than she is showing during the day?
Those times (refusals or anxiety dreams) had me wondering whether I should call the whole thing off? I want this process to feel right to Nica, too. It is not just about the goal (being diaper-free) it is also about how we get there and if she is struggling or I am stressed then is this really the right method for us, after all?
But the last two days it has all seemed so natural, so stress-free that it completely feels right again, too. We have got here with no threats, no bribes (cheerios or smarties?) and no shaming for mishaps. We have tried to keep it always as easy-going and 'normal' as possible. I did go through a phase (after a terrible day 5 of the process) of doing a bit of a 'potty party' and doing a (home-made) potty-song every time she peed or pooed in the potty, even the slightest bit… but I am an Alfie Kohn unconditional parenting afficionada and that wasn't quite sitting right with me. So, now I have gone back to supporting the inherent intrinsic motivation for mastering this skill and I simply describe what is happening to her "you felt the urge to pee, you walked over and you peed" and then I rejoice and share in her happiness if she choses to 'celebrate' or I move on with her if she choses to treat it (rightly?) like the most natural thing in the world. We have re-ditched the 'good job'. And the rest of the time there is no pressure. If she says she doesn't need to go I take her word for it – which puts less pressure on her and helps ensure she is in charge of the pace of this process. It is her process after all.
So, it hasn't been smooth going. It hasn't happened easily, in three days… but it is happening. We are diaper-free, she is going to the potty on her own, spontaneously, naturally and of her own volition and overall I am proud of us for making this transition earlier rather than later for her, for her confidence, hygiene and autonomy, for the environment and for us parents, too, as this promises to be easier in many ways in the long run (not least on our wallet). The three-week method is working just fine for us!


text:
You already know that if I was to use only one skin care brand it would be Perricone MD. If I was to use only one make-up brand it would be Charlotte Tilbury. If I was to shop in just one home decor store it would be Anthropologie (with furniture from West Elm, obviously). Sometimes I go to place an online order for Asda delivery and suddenly, without any warning or you know - awareness - I'm on the Anthropologie website putting a floral rolling pin & a set of hedgehog measuring spoons into my basket. The uncontrollable power of the internet. Sometimes though - just sometimes - I think hey, I could re-create that for less than a tenner (looking at anything animal-figure-toy-esque). Or these bold, Greek inspired plant pots - when they were on the website I used to go & admire them almost religiously on a more frequent basis than I'm willing to admit. But they sold out quicker than bread (as everything on Anthropologie) and all I was left with was empty hands. And a heap of inspiration!
What will you need to make these Anthropologie-style hand-painted plant pots?🌿 old plant pots or bowls (I got the smallest one from Wilkos for just £1!)
🌿 spray paint of choice (I'm using Rust-Oleum's Painter's Touch spray paint in Sun Yellow, Cherry Red & Brilliant Blue - see them here)
🌿 white acrylic paint & thin brush
🌿 a bucketful of patience
And how to make them?I'm making it sound more dramatic then it is - these are actually super easy to make! Spray paint your pots - I did 2 coats and also a little bit on the inside, just because some of my planters were a little smaller than the plant pots themselves and the inside rim was peaking through. Once dried (& super glossy - I think the high gloss gives them that expensive look, don't you think?) take your white acrylic paint & a paint brush and start painting the designs. My favourite (and the easiest) was the blue one - it was actually incredibly therapeutic to paint the flowers! Not gonna lie, the red one was the most difficult one and definitely the poorest attempt from my side. Completely lost my nerve on it (then went back to carry on with the blue pot therapy). Now leave to dry & voila you have some swanky Anthropologie pots for way less than a fiver each!
Are you fan of Anthropologie inspired DIY projects?
I have a few more aces up my sleeve so keep your eyes peeled!


text:
The transsulfuration enzyme cystathionine γ‐synthase (CGS) catalyses the pyridoxal 5′‐phosphate (PLP)‐dependent γ‐replacement of O‐succinyl‐L‐homoserine and L‐cysteine, yielding L‐cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine β‐lyase (CBL), and refined at 1.5 Å resolution to a crystallographic R‐factor of 20.0%. The enzyme crystallizes as an α4 tetramer with the subunits related by non‐crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quarternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate‐binding functions of individual active site residues. Enzyme‐substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate‐binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP‐dependent replacement reaction at Cγ of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.
The sulfur‐containing amino acids l‐cysteine, l‐homocysteine and l‐methionine are metabolically linked via the transsulfuration and reverse transsulfuration pathways. In transsulfuration, employing the sequential action of cystathionine γ‐synthase (CGS) and cystathionine β‐lyase (CBL), l‐cysteine condenses with activated l‐homoserine to form the intermediate l‐cystathionine (CTH), which subsequently is split asymmetrically into l‐homocysteine and pyruvate. Homocysteine may be methylated to yield l‐methionine. Different organisms seem to display distinct spectra of transsulfuration enzymes: plants and microbes employ only the forward pathway from cysteine to methionine, in mammals only the reverse transsulfuration is found, and fungi support transsulfuration in both directions.
CGS (EC 18.104.22.168), encoded by the metB gene, catalyses the first step in transsulfuration, which at the same time can be regarded as the first specific step in l‐methionine biosynthesis, i.e. a pyridoxal 5′‐phosphate (PLP)‐dependent γ‐replacement leading to the formation of CTH from a homoserine ester and l‐cysteine. CGS has been purified from a variety of bacteria and plants, including Salmonella typhimurium (Kaplan and Flavin, 1966b), Escherichia coli (Tran et al., 1983), Arabidopsis thaliana (Ravanel et al., 1998), wheat (Kreft et al., 1994) and spinach (Ravanel et al., 1995). While it has been found that the enzymes from prokaryotes and eukaryotes use different substrates, i.e. O‐succinyl‐l‐homoserine (OSHS) and O‐phospho‐l‐homoserine, respectively, in vitro, the plant enzymes also accept the non‐physiological esters, albeit with a 10‐fold higher Km (Datko et al., 1974; Ravanel et al., 1995). In all cases investigated, active CGS has been identified as a tetramer of identical or closely related 40‐50 kDa subunits with one PLP cofactor bound per monomer via a Schiff base linkage to an active site lysine (Kaplan and Flavin, 1966b). In the E.coli CGS, PLP is covalently linked to Lys198 (Martel et al., 1987) and gives rise to a strong absorption of the holoenzyme at 422 nm and a weaker absorption at 325 nm, originating from the ketoenamine and enolimine forms of the Schiff base, respectively. The sequences of the enzymes from different kingdoms are ∼30% identical, and the highest homologies are found for the ∼12 residues comprising the consensus PLP‐binding site (Fearon et al., 1982).
Steady‐state kinetics suggest that CGS employs a ping‐pong mechanism, commonly encountered in PLP‐dependent enzymes (Litzenberger Holbrook et al., 1990), with a slightly alkaline pH optimum (Kaplan and Flavin, 1966a; Litzenberger Holbrook et al., 1990). Besides the replacement, which is the only relevant reaction in vivo, CGS in the absence of l‐cysteine catalyses in vitro a γ‐elimination, breaking up OSHS into α‐ketoglutarate, succinate and ammonia, as well as a variety of other non‐physiological reactions (Guggenheim and Flavin, 1969b). Both chemical intuition (requirement for the elimination of the γ‐substituent in either route) and pre‐steady‐state kinetic analyses (absorbance maximum at 300 nm, appearing concomitantly with the bleaching of the 422 nm absorbance of the holoenzyme) suggest a common partitioning intermediate in the replacement and elimination reactions, presumably an α‐imino β‐γ‐unsaturated pyridoxamine derivative (Brzovic et al., 1990).
The enzymes involved in methionine biosynthesis are interesting targets for the development of antibiotics and herbicides. Our group has therefore set out to acquire high resolution structural data for these proteins in order to elucidate their reaction mechanisms and modes of interaction with inhibitors. Recently, the structure of E.coli CBL, the enzyme which catalyses the second step in the methionine pathway, has been solved at 1.82 Å resolution, both alone and in complex with the inhibitors aminoethoxy‐vinylglycine (AVG) and trifluoroalanine (Clausen et al., 1996, 1997a). CGS exhibits 37% similarity (29% amino acid identity) to CBL, and a common evolutionary origin for the two enzymes has been suggested (Belfaiza et al., 1986). We subsequently have been able to purify recombinant E.coli CGS (386 residues, 40 kDa) in large amounts and have obtained well diffracting single crystals (Wahl et al., 1997). The initial analysis of the diffraction data of one crystal form (space group P1) indicated the presence of two independent CGS tetramers per asymmetric unit, both of which exhibited local 222 symmetry. Herein, we describe the crystal structure of the native enzyme in a newly discovered crystal form with space group C2, refined at 1.5 Å resolution, and discuss its implications for the specificity and mode of action of CGS.
Results and discussion
Quality of the model
Because the structures of the enzyme in the two different space groups are almost identical, we only refer to the higher resolved C2 structure in this and the following discussions. Accordingly, the refined model comprises one CGS homotetramer, four covalently bound PLP molecules, 1301 waters oxygens and one 2‐methyl‐2,4‐pentanediol (MPD) molecule, and maintains small deviations from standard geometry (Table I). The final crystallographic R‐factor and free R‐factor (Rfree) are 20.0 and 25.5%, respectively, for the diffraction data between 8.0 and 1.5 Å. The mean positional error of the atoms as determined by a Luzatti plot (Luzatti, 1952) is 0.19 Å. Especially for the β‐strands, the internal side chains and the region around the cofactor, the error is likely to be lower. The main chain dihedral angles of all non‐glycine residues are within energetically allowed regions of the Ramachandran plot (Ramachandran and Sasisekharan, 1968), except for two serine residues (Ser326 and Ser178); 89.7% of the amino acids lie in the most favoured area of this plot. Both outlying serine residues are integral parts of the active sites and their conformation is determined unequivocally by the excellent electron density in these regions of the tetramer. A 2Fo‐Fc map contoured at 1.2σ shows continuous density for all main chain atoms with the exception of two flexible residues at the N‐terminus. Only the side chain of a single residue, Glu40, which is positioned on the surface of the molecule, displays no electron density.
Monitoring of the free R‐factor, model building into non‐crystallographic symmetry (NCS)‐averaged density and incorporation of a round of simulated annealing should have eliminated the model bias introduced from the CBL structure. The general correctness and quality of the solution was also attested by the 'omit' density which appeared for the PLP cofactor and improved continuously during the refinement (Figure 1).
All significant electron density patches were accounted for by the refined model, except for a small region which was symmetrical about the crystallographic 2‐fold axis and appeared in the late stages of the refinement. According to the composition of the crystallization buffer, it most probably corresponds to a MPD molecule. The co‐crystallization of this molecule is presumably one of the determinants of the change in space group from P1 to C2.
Overall structure of the CGS monomers
Secondary structural elements were assigned on the basis of characteristic hydrogen‐bonding patterns and φ/ψ backbone torsion angles [DSSP (Kabsch and Sander, 1983), O (Jones et al., 1991)]. Thereafter, the enzyme consists of 44.2% helical structure, 22.5% β‐bends, 17.7% β‐sheets and 15.6% unclassified coil structure. The short helix 7 comprising residues 184‐188 exhibits a hydrogen‐bonding pattern typical for 310 helices.
Like most other PLP enzymes, each CGS subunit can be divided into three domains (Figure 2). (i) Each subunit has an extended N‐terminal domain (residues 1‐51) composed of helix 1 and an extended loop structure comprising 38 residues. (ii) The large PLP‐binding domain (residues 52‐247) with an open, mainly parallel, seven‐stranded β‐sheet (β‐strands a, g, f, e, d, β and c with directions +, −, +, +, +, + and +, respectively) at its centre, which forms a curved plane around helix 3. All β‐strand cross‐overs are right‐handed. The β‐sheet in the PLP‐binding domain is sandwiched between seven helices; helices 2, 5, 6 and 7 are located on the solvent‐accessible side of the sheet, helices 3, 4 and 8 lie on the opposite side and are involved in the formation of the intersubunit and interdomain interface. At the latter interface, PLP is covalently attached via a Schiff base linkage to Lys198, located near the N‐terminus of helix 3, and the tails of strands d, e and f. The PLP‐binding domain is connected by the long α‐helix 9 to the C‐terminal domain. The connecting helix consists of 34 amino acids and has a kink near Thr247, adopting a characteristic structure which is also found in CBL (Clausen et al., 1996). (iii) The central part of the C‐terminal domain (residues 248‐385) is a five‐stranded, mainly antiparallel β‐sheet with a topology in Richardson notation (Richardson, 1981) of +1, +3X, −1X, −1. The formation of a five‐stranded β‐sheet in the C‐terminal domain has not been observed in any other related PLP enzyme structure. The cross‐overs are right‐handed, and helices 10, 11, 12 and 13 of the C‐terminal domain are all located on the solvent‐accessible side of the β‐sheet.
Quarternary structure and crystal packing
The crystal structure reveals the presence of a homotetramer with local 222 symmetry. The NCS axes could be identified unequivocally in a locked self‐rotation search performed with GLRF (Tong and Rossman, 1990) and were exploited in early stages of the model building. The crystal structure is in accord with the native tetrameric state of CGS in solution (Tran et al., 1983). The quarternary assembly displays approximate dimensions of 90×85×75 Å (Figure 2). Despite their crystallographic independence, any pair of monomers (AB, AC and AD) can be superimposed with r.m.s. deviations of 0.276, 0.283 and 0.241 Å, respectively.
The CGS monomers are oval, beetle‐shaped molecules of ∼50×55×70 Å. The compact monomer shape is achieved by packing the PLP‐binding domain against the C‐terminus, while an N‐terminal extended loop protrudes from the bulk of the subunit, forming a clamp to the neighbouring monomer. The clamping occurs mainly through hydrophobic interactions: Phe35 and Phe38 are bound to a hydrophobic pocket on the C‐terminal domain of the second subunit, which is lined with residues Leu311*, Leu315*, Leu323*, His 336* and Met340* (the asterisks indicate residues from the second subunit of the active dimer). These interactions enable residues 42‐50 to come in close contact with the neighbouring active site, and to participate in cofactor binding and formation of the active site entrance. Furthermore, the guanidinium group of Arg114* of the second monomer docks via hydrogen bonds to three carbonyl oxygens to the C‐terminus of helix 3 (PLP phosphate‐binding helix). With its N‐terminus binding the PLP phosphate, helix 3 effectively mediates an intersubunit salt bridge between the two peripheral ionic groups via its helix dipole. Overall, it can be seen that an intricate network of interactions orchestrates contributions from residues of all three domains of a monomer and from the two subunits of the active dimer to both symmetrical active sites.
Within the homotetramer, the four monomers clearly assemble into two catalytically active dimers (AC and BD, Figure 2), engaging in extensive intradimer contacts and in a smaller number of interdimer contacts. The three different monomer‐monomer associations bury 5127 Å2 of the solvent‐accessible surface in the tight (AC) connection, and 2288 Å2 (AB) and 3369 Å2 (AD) in the weaker connections, as calculated with GRASP (Nicholls et al., 1991). The monomer‐monomer interactions within an active dimer encompass 32 hydrogen bonds, two salt bridges and various hydrophobic contacts, while the dimer‐dimer interactions include 76 hydrogen bonds and 12 salt bridges, rendering the dimer‐dimer interface highly polar. Only in the centre of the tetramer, there is a striking cluster of hydrophobic residues which seems to play a pivotal role in the stabilization of the quarternary assembly. One phenylalanine (Phe236), one isoleucine (Ile27) and two leucines (Leu29 and Leu240) from each monomer come together entertaining van der Waals and stacking interactions. The intimacy of the contacts is protected by the shielding effect of peripheral Tyr239 residues and salt bridges between Arg243 and Asp205.
The above quarternary association leads to the formation of four active sites per tetramer, two made up within each active dimer. These two active sites are quite close to each other (P‐P distance of the PLP cofactors = 21.4 Å). For several enzymes of the γ‐family, evidence has been obtained suggesting that only one active site per dimer is actually operating or being inhibited by mechanism‐based inactivators (Abeles and Walsh, 1973; Silverman and Abeles, 1977; Johnston et al., 1979). Interestingly, the present structure reveals a corresponding asymmetry with respect to the B‐factors for residues 34‐50, which are thought to be essential for substrate binding. For the monomers of one of the active dimers (BD), this region is significantly more disordered (average B‐values 65 Å2 versus 40 Å2 in AC). The more flexible active site environment could lead to an easier acceptance of the substrates via an induced‐fit mechanism.
Active site
The active site design is shown schematically and spatially in Figure 3. The boundaries which enclose the cofactor are made up of the N‐terminal ends of helices 3 and 4, the C‐terminal tails of strands d, e and f, and their associated loops. The opposite border of the active site lining the bound substrate is constructed by the C‐terminal loops of strands C and D of the C‐terminal domain, and an N‐terminal segment (44*‐50*) of the neighbouring subunit.
Continuous electron density shows that the aldehyde functionality of the uncomplexed PLP and the ϵ‐amino group of Lys198 have condensed to form the internal aldimine (Figure 1). The nitrogen of this Schiff base is protonated as deduced from the absorption maximum of the CGS crystals at 425 nm. The positive charge is stabilized by interaction with the deprotonated PLP hydroxyl group at C3.
Besides the covalent bond, the cofactor is anchored predominantly in the active site through its phosphate group, for which seven hydrogen bonds to protein residues are discernible, i.e. to the main chain amide nitrogens of Gly76 and Met77, and to the side chains of Ser195, Thr197, Tyr46* and Arg48*. The latter hydrogen bonds are strengthened by charge‐charge interactions. Binding of the PLP phosphate is improved further by its interaction with the positive end of the helix 3 macrodipole, as mentioned above. The OP4‐C5′‐C5‐C4 torsion angle exhibits an energetically favourable 84°, fixing the ester oxygen at the A‐face (Ford et al., 1980; in the direction of the protein interior) of the cofactor.
The PLP pyridine is sandwiched between Tyr101 and Thr175/Ser195 which impede any vertical movements of the cofactor with respect to the pyridine ring plane. The latter two residues are in van der Waals contact (shortest distances to PLP atoms 3.8 and 3.7 Å, respectively) with all atoms of the cofactor ring system. The phenol ring of Tyr101 is located 3.7 Å above the PLP pyridine ring with a tilt angle of ∼10° between the best planes through both rings. Apart from restraining the cofactor, the resulting ring‐stacking interactions should, in analogy to the situation in aspartate aminotransferase (Hayashi et al., 1990), increase the electron‐sink character of the cofactor. The hydroxyl group of Tyr101 is positioned close to the guanidinium group of Arg48* (3.1 Å) and to the positively charged nitrogen atom of the internal aldimine (4.4 Å). It can therefore be inferred that Tyr101 exists as a phenolate in the pH range of 8.2‐9.5, over which the CGS activity is maximal.
Asp173 constitutes another catalytically important residue. It builds a strong hydrogen bond/salt bridge (2.71 Å) to the PLP pyridine nitrogen (N1), thereby stabilizing its positive charge and increasing the electrophilic character of the cofactor. As a component of an extensive hydrogen‐bonding network (Figure 3A), the carboxylate group of Asp173 is fixed in the geometrically optimal position for the contact to N1. Furthermore, the hydrogen bonds which originate from Asp173 should permit charge dissipation during the catalytic cycle into the protein periphery.
Beside Lys198, Asp173 and the structurally important Gly76, Arg361 is largely conserved in the α‐ and γ‐families of PLP‐dependent enzymes, with the exception of those members whose substrates miss the α‐carboxylate group. Accordingly, this residue is assumed to bind the α‐carboxylate group of the incoming substrate. The orientation and position of the Arg361 side chain is fixed in the present structure by interaction with the amide oxygen of Asn148. In the CGS active site, two water molecules (O597 and O659) are found in the vicinity of the guanidinium group of Arg361 (Figure 3). The locations of these water molecules could point to the positions of the substrate α‐carboxylate oxygens.
The C2′‐methyl group and the OH3′‐hydroxyl group of the cofactor are not involved in any specific interactions with the protein. At the corresponding side of the cofactor, the binding pocket has a significantly hydrophobic character which originates from the apolar residues Phe176 and Leu327. Only one weak hydrogen bond is formed between the deprotonated hydroxylic group at C3 (OH3′) and an active site water molecule (O3). In contrast, aminotransferases provide a multitude of polar interactions with OH3′. These interactions are believed to be of importance in lowering the pKa of the internal aldimine (Yano et al., 1993) and for binding an incoming water molecule for ketimine hydrolysis (John, 1995). These mechanistic features are not relevant for CGS. Furthermore, limited hydrogen bonding to OH3′ is not opposing reorientation of the PLP pyridine ring during transaldimination and should, in analogy to the Y225F mutant of aspartate aminotransferase (Goldberg et al., 1991), result in a decreased Km for substrates and inhibitors.
Comparison with CBL
The three‐dimensional fold of CGS is related to those of the members of the α‐family of PLP‐dependent enzymes (for an overview, see John, 1995) and, of course, to the evolutionarily related γ‐family enzyme CBL. For comparison of the respective active sites, residues Tyr101, Asp173, Lys198, Ser326, Arg361 (CGS nomenclature) and the PLP of CGS and CBL were used to guide a least squares superposition with the program O (Jones et al., 1991). The resulting overlays of the Cα traces and of selected active site residues are shown in Figure 4.
Like CGS, CBL is a homotetramer in solution (Belfaiza et al., 1986), with very similar shape, dimensions, relative orientation of subunits, and domain structure. The least squares superposition aligns 340 Cα atoms with an r.m.s. deviation of 1.482 Å. All secondary structural elements of the PLP‐binding and most of the C‐terminal domain superimpose almost perfectly. Interestingly, even the N‐terminal residues 1‐35 of CBL and CGS fold similarly, although they exhibit no sequence homology.
The two main regions of divergence in the overall structures of CGS and CBL (referred to as I and II) are indicated in Figure 4A and B. Region I (CGS 336‐358, CBL 349‐369) is situated in the C‐terminal domain forming an extended loop including helix 12. For CGS, this loop protrudes much further into the active site than for CBL, considerably narrowing the active site cleft at this border. The arrangement of this loop furthermore leads to a reorientation of helix 12 by 90°, thereby opening a second entrance to the active site. This stretch of the protein contains no catalytically important residues and no amino acids implicated directly in substrate binding. Rather, by influencing the overall shape and the electrostatic curvature of the active site channel, this region presumably plays a steric role for substrate binding or product release.
The second region (region II) is composed of residues 41‐44 in CGS and 40‐54 in CBL, and constitutes the area of predominant differences between the two enzymes. In CGS, it is again arranged in a short loop, whereas in CBL it folds in an additional helix of the N‐terminal domain which protrudes into the second subunit of the active dimer. Here it builds up the active site wall near the phosphate group of the cofactor and carries two arginines (Arg47* and Arg49* in CBL nomenclature) which were proposed to be important in CBL for binding the distal carboxylate group of cystathionine (Clausen et al., 1997b).
In the superposition of the active sites of CGS and CBL (Figure 4C), the catalytically important residues Lys198, Tyr101 and Asp173 align almost perfectly. Even the hydrogen‐bonding network around Asp173 is quite conserved. Similarly, the positions of residues involved in the binding of the PLP phosphate group (Tyr46*, Arg48*, Gly76, Met77 and Thr197) are closely related. Nevertheless, we observe some functionally significant differences between the two structures.
Probably the most interesting amino acid exchanges refer to the CBL Tyr338 and Phe55*, which are mutated to Glu325 and Asp45* in CGS. In the structure of the CBL‐AVG complex, the phenolic rings of Tyr338 and Phe55* engage in hydrophobic interactions with the inhibitor and enclose it in the active site. The corresponding regions of CBL previously have been proposed to determine the permissible chemical nature of the substrate and its orientation (Clausen et al., 1997a). The change from tyrosine/phenylalanine to glutamate/aspartate corresponds to an exchange of hydrophobic to acidic side chains, which can be correlated with the enzymes' substrate specificity. Accordingly, the CBL substrate should be bound in analogy to AVG via hydrophobic interactions, while the second CGS substrate, cysteine, is more appropriate for an interaction with the acidic side chains of glutamate and aspartate.
A shift in the locations of the side chains of Tyr46* (CGS) and Tyr58* (CBL) leads to a much closer approach of the PLP phosphate and the tyrosine side chain in CGS. By this rearrangement, the side chain of Tyr46* adopts an orientation different from that of the ϵ‐amino group of Lys198 which could be of catalytic importance (see below). A further minor difference between CGS and CBL is the change from Trp340 (CBL) to Leu327 (CGS), which should have little influence on catalysis or substrate binding. The position, which can be occupied by a phenylalanine, a leucine or a tryptophan, is not strictly conserved in the γ‐family of PLP enzymes and shows a variation which seems to be independent of the catalysed reaction type or the converted substrate.
Modelling of substrate‐binding modes and reaction mechanism
CGS represents the only case of a PLP‐dependent enzyme that catalyses a replacement or elimination at the γ‐carbon of an amino acid substrate. The present X‐ray structure in combination with molecular modelling of enzyme‐substrate complexes suggests a likely catalytic mechanism for CGS. This mechanism is based, with some slight modifications, on a mechanism proposed by Brzovic et al. (1990).
OSHS binding to the CGS enzyme has been modelled in the state of the external aldimine formed between the OSHS molecule and the PLP cofactor. The PLP moiety and the α‐carboxyl group were pre‐oriented according to the binding mode of AVG in the inhibitor complex crystal structure of the related X‐ray structure of CBL (Clausen et al., 1997a), with the α‐carboxyl group in hydrogen‐bonding contact with Arg361. The carboxyl group of the O‐succinyl part of the molecule was oriented in hydrogen‐bonding distances to Arg48* and Arg106. The situation before and after energy minimization is depicted in Figure 5. The external aldimine‐enzyme complex minimized to a total energy of −901 kcal/mol. The minimization brings the α‐carboxyl group of the external aldimine into hydrogen‐bonding distance to Asn148, maintaining the contact to Arg361. The distal carboxyl group of theO‐succinyl moiety remains hydrogen bonded to Arg48* and additionally acquires an H‐bond with Tyr101. A remarkable rearrangement of side chains in the active site occurs as a consequence of the formation of the external aldimine which has also been observed in the CBL‐AVG complex structure (Clausen et al., 1997a). The side chain of Tyr101 undergoes a considerable rotation and comes to lie parallel to the pyridine ring of the external aldimine. The side chain of Arg106 moves away from the O‐succinyl‐carboxyl, breaking the initial hydrogen bond.
Figure 6A shows a solid surface/electrostatic potential representation of the active site, calculated without the OSHS external aldimine, plus its energy‐minimized model. The blue positive recognition regions for the two OSHS carboxyl groups are due mainly to the side chains of Arg361 and Arg48*. The negatively charged recognition site for the α‐amino group of OSHS is not visible on this surface because it arises mainly from the deprotonated OH3′ of the PLP which is buried in the cofactor‐binding pocket. The charge distribution in the right well at the entrance of the channel is determined by Arg49* and Asp45*, and that of the lower part of the entrance by Glu325. In the energy‐minimized complex (surface not shown), the side chain of Glu325 moves into hydrogen‐bonding contact with Arg361 (Figure 5). Thus, the carbonyl function of OSHS is no longer close to Glu325 and the negative surface potential of the latter residue is partly neutralized by the formation of the Arg361‐Glu325 interaction.
CGS catalyses the γ‐synthesis of CTH from OSHS and l‐cysteine and, therefore, has to adopt two different substrate molecules. In accordance with substrate modelling, OSHS would occupy the main part of the binding pocket, extending from Arg361 to a very basic region lined with Arg106, Arg48* and Asn227* (Figure 6A). With OSHS fixed in this orientation, there is a second potential binding site which is electrostatically optimally designed for hosting a zwitterion and could, therefore, be responsible for binding l‐cysteine. In Figure 6B, the leaving group of OSHS has been omitted and the second substrate, l‐cysteine, has been docked manually with the α‐amino and α‐carboxy groups in hydrogen‐bonding contacts with their putative recognition functions Asp45*/Glu325 and Arg49*, respectively. In this model, the l‐cysteinate sulfur is arranged perfectly for a nucleophilic attack at the Cγ of the homoserine‐PLP derivative. In contrast to CGS, the active site entrance of CBL is very narrow owing to the location of helix 3, which is absent in CGS and which covers the proposed cysteine‐binding site like a lid (Figure 6B). In detail, the side chain of Arg59* of CBL is approximately in the region of Arg49* of CGS, Phe55* (CBL) in place of Asp45* (CGS), Tyr338 (CBL) in place of Glu325 (CGS) and the other visible residues of helix 3 of CBL cover empty space in CGS. Since all catalytically important residues of CBL are conserved in CGS, we therefore propose that the different chemical reaction types supported by the enzymes are solely a consequence of their different substrate‐binding properties.
As may be expected for this unique and unusual reaction, the reaction mechanism entertained by CGS is rather complicated. It has been much discussed in the literature (Brzovic et al., 1990; and references therein) and appears to involve three residues that act as acid‐base catalysts: (i) for the transaldimination reaction; (ii) for the 1‐3 prototropic shift; and (iii) for protonation of the OSHS leaving group. Based on the structure, we can propose specific residues for each of these functions (Figure 7).
(i) Transaldimination. A scenario similar to that proposed for CBL should hold true (Clausen et al., 1996). After productive binding of OSHS through residues Arg48*, Tyr101 and Arg361, the amino group of the substrate must be deprotonated for the nucleophilic attack on C4′ of the internal aldimine. Y101 should exist in the unliganded enzyme at pH 8.5 (optimal pH, Litzenberger Holbrook et al., 1990) as phenolate due to the two neighbouring positive charges (Arg48*, NH of the internal aldimine). Thereafter, Tyr101 abstracts a proton from the incoming substrate and initiates transaldimination (Figure 7I).
(ii) Generation of the ketimine intermediate as identified in time‐resolved rapid scanning spectroscopy (Brzovic et al., 1990). After transaldimination, Lys198 is responsible for the proton transfer from Cα to C4′ of the cofactor. Due to the absence of the typical quinonoid intermediate, as deduced from the missing characteristic 490 nm absorption band during the course of the reaction (Karube and Matsushima, 1977; Kallen et al., 1985), the proton should be more or less directly transferred from Cα to C4′ as indicated in Figure 7II and III. As argued for aspartate aminotransferase (Kirsch et al., 1984), Tyr46* should have an effect in guiding the amino group of Lys198 during catalysis. In comparison with the corresponding CBL Tyr58*, which is important mainly for cofactor binding, Tyr46* in CGS has moved deeper into the protein interior (see above), thereby favouring a positioning of the protonated Lys198 amino group near C4′ after the initial α‐proton abstraction. From a structural point of view, the side chain of Lys198 can swing like a 'liana' to either Cα, C4′ or Cβ of the substrate. It is therefore also proposed to be the residue abstracting a proton from Cα to initiate γ‐cleavage (Figure 7IV). In the PLP ketimine derivative, the latter atom should have a slightly acidic character (Brzovic et al., 1990), facilitating this proton abstraction process.
(iii) Release of succinate. To gain insight into this part of the reaction, it would be helpful to elucidate the structure of the CGS‐propargylglycine (PG) complex. Unfortunately, the CGS crystals suffer severely when treated with PG and lose much of their diffraction power. Inactivation by PG is the consequence of the covalent linking of the PG‐Cγ to an active site nucleophile other than Lys198. For the closely related cystathionine γ‐lyase, it has been reported that this residue should be either a cysteine or a tyrosine (Washtien and Abeles, 1977). In accordance with substrate modelling experiments and the structure of the CBL‐AVG inhibition complex (Clausen et al., 1997a), Tyr101 seems to be the corresponding residue in CGS. Thereafter, Tyr101 facilitates release of succinate in the physiological reaction as proposed in Figure 7.
The resulting β‐γ unsaturated ketimine (Figure 7VI) exhibits a pronounced electron deficiency, caused by the protonated Schiff base, leading to activation of Cγ towards Michael nucleophilic addition by l‐cysteinate. The last steps of the catalytic cycle (VII‐IX) are the reverse of the previous steps including Cβ protonation, C4′ deprotonation and Cα protonation. Reverse transaldimination of the external product aldimine releases CTH into the active site and completes the net γ‐replacement reaction.
In summary, the CGS structure, in comparison with that previously determined for the related CBL, illustrates how versatility can be conferred upon a cofactor by the intricate design of the active site of the corresponding enzymes. Specificity in the present example presumably stems from the steric and electrostatic (in)accessibility of the active sites rather than the exchange of catalytically important residues. High resolution structures, as exemplified by the present work, should prove instrumental in the design of specific inhibitors of transsulfuration components, which may be an important lead to novel herbicides and antibiotics, acting through interference with microbial and plant methionine biosynthesis.
Materials and methods
Crystallization and data collection
Recombinant CGS was purified and crystallized as described previously (Wahl et al., 1997). For initial data collection, the largest crystals were soaked in cryoprotectant (reservoir buffer supplemented with 15% MPD) and mounted in a liquid nitrogen stream (KGV, Karlsruhe, Germany), which allowed data collection over prolonged times at 113K. Diffraction data initially were collected on our in‐house MAR‐Research (Hamburg, Germany) image plate with graphite‐monochromated CuKα radiation (λ = 1.5418 Å) at 50 kV/100 mA from a Rigaku (Tokyo, Japan) RU 200 rotating anode. A single specimen produced a data set complete to 2.4 Å resolution in a 180° φ‐scan with a 1.0° frame width and 1600 s exposure per frame. The data were processed as before (Wahl et al., 1997) with the program packages MOSFLM (Leslie, 1991) and CCP4 (Collaborative Computational Project, 1994).
Initially, only crystals which exhibited space group P1 with >25 000 non‐hydrogen atoms per asymmetric unit were characterized, necessitating the acquisition of a larger number of reflections to allow independent refinement of all asymmetric atoms. At times, crystals appeared under the same reservoir conditions which exhibited a distinct, compact morphology (∼0.3×0.3×0.3 mm3). One of these crystals yielded data beyond 1.5 Å resolution at an X‐ray wavelength of 1.1 Å at the Deutsche Elektronen Synchrotron, beamline BW6, Hamburg, Germany at 100K (Oxford Cryosystems, Oxford, UK). The data were processed as before and indicated space group C2 with a = 160.04 Å, b = 61.30 Å, c = 153.84 Å, and β = 104.18°, suggesting a single CGS tetramer per asymmetric unit. The statistics of this data set are summarised in Table I.
Structure solution and refinement
The structure of CGS initially was solved by molecular replacement with a P1 data set and later transferred to the higher resolution C2 data. In all structure solution and refinement routines, only data with F ≥2σ(F) were used (Table I), 10% of which were set aside for the calculation of an Rfree. Best‐fit alignment (Genetics Computer Group, 1997) of the sequences of E.coli CGS and CBL revealed homologies between the enzymes, based on which a model for rotation‐translation searches was constructed: all identical residues were retained, the others were replaced with alanines, the extended N‐terminus of CGS (residues 1‐50) was omitted completely, and all B‐values were reset to 20 Å2.
Using the P1 data up to 3.5 Å resolution, the partial polyalanine model, assembled into a tetramer guided by the CBL structure, yielded two solutions in a molecular replacement search in AMoRe (Navaza, 1994) well above the background, with a combined R‐factor and correlation coefficient of 48 and 35%, respectively. While the resulting 2Fo‐Fc electron density map was not easily interpretable, a map after 8‐fold cyclic averaging with RAVE (Kleywegt and Jones, 1994), using NCS operators extracted with LSQMAN (Kleywegt, 1996), allowed unequivocal main‐ and side‐chain tracing in the central parts of the molecule. Refinement, model rebuilding and water incorporation proceeded smoothly via rigid body, positional and later B‐factor optimizations in X‐PLOR (Brünger, 1992) using the parameters developed by Engh and Huber (1992), and employing weak NCS restraints. During this process, the R‐factor and the Rfree dropped from 48 to 28% and from 49 to 33%, respectively.
Resetting the B‐factors to 20 Å2, one tetramer model at this stage of the refinement was used in rotation‐translation searches with the C2 data set truncated at 3.5 Å resolution, allowing the positioning of a CGS tetramer in the new space group (R‐factor = 30%, correlation coefficient = 55%). The unequivocal solution displayed clear electron density for the PLP cofactor which was not included in the initial search model. All data to 1.5 Å resolution were included stepwise in rigid body refinements (X‐PLOR). Further improvement of the model was achieved in positional and B‐factor optimizations (X‐PLOR). One round of simulated annealing (Weis et al., 1990) was included to minimize the model bias. Water molecules were placed into the electron density with the routine ARP (Lamzin and Wilson, 1993) and manually checked for their authenticity. In the last round of the refinement, an anisotropic overall B‐factor refinement was applied. The final model contained 12 993 non‐hydrogen atoms and converged at an R. Rfree of 20.0%/25.5%. The atomic coordinates and experimental intensity data have been submitted to the Brookhaven Protein Data Bank (accession No. 1CS1).
Molecular model building
Molecular modelling was pursued with modules Viewer, Builder, Docking, Delphi and Discover3 of program Insight II (Version 97.0; Los Angeles, 1997). The dimer consisting of subunits A and C of the refined CGS X‐ray structure was chosen as the starting point for the model building and energy minimization of the modelled complex. OSHS, and subsequently its external aldimine with PLP, were generated and positioned into the active site following the binding of AVG in CBL. The Consistent Valence Forcefield (CVFF) of the Insight II program was used for the energy minimization with Discover3 which was run over 2000 minimization steps until convergence (0.1 kcal/mol tolerance). The protein dimer and the ligand molecule were minimized simultaneously.
Generous financial support from a stipend of the 'Peter und Traudl Engelhorn Stiftung zur Fürderung der Biotechnologie und Gentechnik' to M.C.W. is gratefully acknowledged.
- Copyright © 1998 European Molecular Biology Organization


text:
With a surprisingly strong vote, 53 percent of Arkansas's voters said last Nov. 8 that they wanted to bring medical marijuana to the state. This creation of a medical marijuana program via vote of the people came over the strong objections of Governor Hutchinson, most all of the state's other elected officials and those charged with implementing health policy in the state. In the aftermath of the election, while most (including Hutchinson) have fairly gracefully accepted the will of the people, a few political leaders continue to resist medical marijuana at every turn.
Over two dozen bills have been introduced in the current legislative session to amend the constitutional amendment in large and small ways. The amendment itself did allow the legislature (by a two-thirds vote in both houses) to reform the program as long as core key provisions — the right for those with certain medical conditions to use marijuana without fear of prosecution and the number of dispensaries and cultivation sites — were not touched. Some of the bills that have been introduced are only intended to implement the program in a more methodical manner than at the breakneck pace laid out in the amendment, providing more time for the commission established to run the program a few more weeks to get rules and regulations finalized. Indeed, it's good public policy to get a major new program done correctly on the front end.
Other legislation — mostly carried by Rep. Doug House (R-North Little Rock), who was tasked with leading the charge on medical marijuana's implementation — simply fleshes out elements of what was already a fairly detailed amendment. One bill that is already law clarifies doctors' role in the process as a fairly limited one in which they simply confirm that the individual has been previously diagnosed with one of the qualifying conditions. Other legislation that will be passed creates new licensing requirements for those who move pot from a cultivation facility to a dispensary. Finally, new legislation will regulate the advertising of medical marijuana. (The quote of the legislative session by House: "We don't want a dancing bear and flying rabbits and stuff on the advertising that would cause kids to want to frequent these establishments."). While the ban on advertising veers into choppy constitutional waters because medical marijuana is now a legal product in Arkansas, this set of bills does little to undermine availability or use of medical marijuana.
Other legislation, however, proposes to more fundamentally undermine the intent of the medical marijuana program. These include a proposal to delay implementation until the federal government changes its policy toward medical marijuana (currently illegal, of course). Another bans the smoking of marijuana, meaning that marijuana could be delivered only by edibles. To close that loophole, another bill would ban edibles except in rare cases.
A new Talk Business & Politics/Hendrix College poll released this week indicates that Arkansans meant it when they voted for medical marijuana last year, as majorities of the state's voters oppose these efforts to undermine implementation. The survey examined attitudes about the proposals to delay implementation until federal legalization and the ban on smoking. On both issues, while about the same percentage of voters who favored the measure oppose intrusions into it, younger voters, men, African Americans and political independents were most thoroughly opposed to the provisions not included in the original amendment.
There is one more outstanding piece of legislation that may create the most interesting battle of the session in the area of medical marijuana. House Bill 1460 would protect employers from lawsuits if they took action, including termination, against employees who violated a substance-free workplace but were qualified users of medical marijuana. This legislation would seem to bump up against the commitment in the amendment that users may not be discriminated against in the workplace. With strong backing from business interests in a business-friendly legislature, HB 1460 could well get the required supermajority. As shown from the flurry of action in both the legislature and the Medical Marijuana Commission, medical marijuana in Arkansas has already been a boon for the lobbying industry. It may also soon begin to benefit lawyers as laws that may be in conflict with it are tested in court.
During the state's century-long experimentation with direct democracy, Arkansas's voters have occasionally done good with the power they have to create policy at the ballot box (modernizing the state's courts and county government) and just as often done ill (barring same-sex couples from access to marriage and the state's public education institutions from teaching evolution). But, as shown in the aftermath of the creation of a complicated (and imperfect) medical marijuana program through Issue 6, the voters are strong believers that their will should be respected. Politicians who ignore that may well pay a price.


text:
October 30, 2005.
Israel 21C, CA.
For 65-year-old Phillip A., sitting and doing the daily crossword puzzle was one of the day's pleasures. But a couple years ago, his vision began to blur at a steady pace, and not only was he unable to read the paper, he also had to stop driving.
Phillip is one of the over 8 million Americans who suffer from age-related macular degeneration (AMD) - a disorder of the central retina, or macula, which is responsible for detailed vision that controls important functional visual activities like reading, recognizing faces, and watching television.
According to the National Eye Institute over 1.7 million Americans over age 50 suffer mild to profound vision loss from advanced AMD, which culminates as end-stage AMD. Patients affected in both eyes often experience a loss of independence, social interaction, and have difficulty with activities of daily living requiring detailed vision.
Until now, older people like Phillip who are afflicted with the untreatable end-stage form of AMD have not had much hope. But thanks to a device called the Implantable Miniature Telescope (IMT) invented by Israeli doctor Isaac Lipshitz, not only can Phillip regain hope, but he also has a clearer future in sight.
VisionCare Ophthalmic Technologies, Inc. - the company Lipshitz and his partner Yossi Gross founded almost 10 years ago - recently completed Phase II/III trials of the IMT in patients with advanced forms of AMD, and the results point to a revolution in the potential treatment of the ailment.
Ninety percent (172/192) of patients in the US-conducted trial met or exceeded the study's protocol-specified visual acuity endpoint: a 2-line improvement in either distance or near best-corrected visual acuity (BCVA). The study protocol required at least 50% of patients achieve this target.
One-year efficacy data from the prospective, multicenter trial showed patients had a mean improvement of over 3 lines on the ETDRS (Early Treatment of Diabetic Retinopathy Study) chart in both distance and near BCVA in the study eye.
In simpler language, the IMT drastically improved the patients' vision, and the results have paved the way for pending approval of the telescope implant by the FDA.
"The trials were conducted at 28 sites in the US, and the results were better than our expectations and better than the FDA's expectations," Eli Aharoni, Visioncare's general manager told ISRAEL21c. "It shows that the IMT can magnify an image up to three times on the retina, and now we have very good evidence of how effective the device is."
According to co-founder Gross, FDA approval is expected by early 2006, and the device will enter the markeplace.
"We already have CE approval for Europe, but we want to launch the device first in the US. We'll soon be going to an IPO as well," Gross told ISRAEL21c.
The IMT, comprised of ultra-precision quartz glass micro-optics, is designed to be a permanent solution for moderate to profound vision loss due to AMD. Smaller than a pea, the telescope is implanted in one eye in an outpatient surgical procedure. In the implanted eye, the device renders enlarged central vision images over a wide area of the retina to improve central vision, while the non-operated eye provides peripheral vision for mobility and orientation.
"At the end of the day, you need to see the full picture," explained Aharoni regarding the one-eye implant. "Even with normal eyes, the focus isn't on the central object first, initially it's on the periphery. It's just like the way we normally look at things. First you look at the bus coming down the street, then you focus in on the number."
It was a chance encounter with a strong focus that first brought Gross and Lipshitz together in 1995 resulting in the telescope's invention.
"I first met Isaac after my son was stung by a bee in his eye. We had to look for a good doctor to operate because it was getting infected. I was referred to him, and he asked me what I did. I told him I developed medical devices, and he said, 'great, there's a problem with AMD - there's no solution. Let's invent one together,'" said Gross.
A serial entrepreneur, Gross has founded 17 other medical device companies, but still serves as consultant to Visioncare.
"There is no cure for AMD. Photo dynamic therapy slows down the deterioration, but until now, there's been nothing to improve the vision. Our telescope doesn't cure AMD either, but we improve the vision. A person can go from almost legally blind to reading fine print," he said.
In the trials, the telescope prosthetic device was implanted in one eye of patients (average aged 76) with moderate to profound visual impairment (BCVA 20/80 - 20/800) in both eyes due to disciform scars (end-stage wet-AMD) or geographic atrophy (advanced dry AMD).
Retina specialists raved about the results.
"This first-of-kind technology is an exciting development for both ophthalmologists and our patients," said Dr. Henry L. Hudson of Retina Centers in Tucson, Arizona. "If approved for use, we would finally have an option for permanent improved central vision and independence for individuals with AMD who have difficulty with their everyday activities and hobbies, or who may feel isolated from society due to their vision."
Hudson presented the safety and efficacy results at the 109th Annual Meeting of the American Academy of Ophthalmology (AAO) and the AAO Retina Subspecialty Day meeting in Chicago.
"Despite the recent advances in the treatment of AMD, there remain a large number of patients who progress to severe vision loss each year," added Dr. Paul Sternberg, M.D., the Chairman of the Vanderbilt Eye Institute, who presented one-year data at the Retina Subspecialty Day meeting. "These patients can experience considerable improvement in visual function and quality of life from this novel device."
As was found in the earlier Phase I study, the device was well tolerated in the eye. Only 11 patients received a standard intraocular lens due to an aborted surgical procedure.
With such impressive results, VisionCare is preparing to make a big splash in the US. In preparation, the company moved its corporate to Saratoga, California but it still maintains f its research facilities in the Tel Aviv suburb of Yehud.
Next month, VisionCare will be one of the featured Israeli companies at the Israel Venture Association's Israel High Tech VC Conference in Santa Clara, California. According to Gross, it was Israeli venture capital that helped the company out of rough times during its development phase.
"We've gone through many designs and stages over 10 years. VC fund Pitango invested in us, and brought in an American CEO - Alan Hill - who has brought the company to a new level," said Gross.
Hill will be speaking at the conference on a Life Science panel in which industry experts will present the future outlook, latest trends, and investment opportunities that are developing in the Life Science field. He could look no further than his own company.
"Nobody has tried and succeeded in implanting a telescope in the eye," said Gross. "Only with a telescope can you have the magnification that eliminates the black spot and restores the vision. We believe that this is the ultimate and only solution for AMD."
Source URL: http://www.israel21c.org/bin/en.jsp?enDispWho=Articles%5El1131&enPage=BlankPage&enDisplay=view&enDispWhat=object&enVersion=0&enZone=Health.
End of article.
Any further reproduction or distribution of this article in a format other than a specialized format, may be an infringement of copyright.
Go to ...
Top of Page.
Previous Page.
List of Categories.
Home Page.
Blind World Website
Designed and Maintained by:
George Cassell
All Rights Reserved.
Copyright Notice
and Disclaimer.


text:
Athena by Heather B. Moore
This volume in the Newport Ladies Book Club focuses on Athena, a single woman from a Greek family who tries desperately to get her married. Athena has never been comfortable with the idea of marriage, and is a commitment phobic, workaholic. Her casual boyfriend wants to move their relationship up to the next level and when Athena resists he accuses her of caring too much about her work. She can't even remember the last time she read a novel, which is proof to him that she needs to reprioritize her life. Athena is stung by his comments and the end of their relationship. To prove to herself that work doesn't have to dominate her life she goes to a used bookstore and meets Grey. Athena is immediately charmed by his kindness and ends up joining a book club advertised in the store. Athena tries to return to life as usual but discovers that she just can't keep away from Grey, she keeps coming up with excuses to visit his shop to see him. They build a tender but fragile romance and then Athena's mother dies unexpectedly in a car accident, leaving Athena responsible to care for her Father with Alzheimer disease. The support of Grey and her new friends from the book club carry Athena through this dark period of her life.
I really loved this book. I thought that the romance between Athena and Grey was so sweet. It was also nice to see Athena grow as a character and resolve some of the issues that held her back from wanting to start a family. It was fascinating to watch Athena discover more about her mother's life and how she had to readjust the judgments she had made of her as a child. I found this conflict so very relatable and I sympathized with Athena as she struggled to find answers about her mother after she died. I really felt for her as she cared for her aging Father and had to figure out what she had to do to take care of him. Overall, I found this to be a very satisfying read, a sweet but thoughtful romance.Visit Heather B. Moore's blog
Visit Heather B. Moore's website
Visit Newport Ladies Book Club Website


text:
Use of unapproved IUDs more widespread
OB-GYN Associates was nailed by the RI Health Department last week for giving its patients non FDA approved IUDs. The products came from Canada and were less expensive than devices approved here in the US, allowing the practices to make a hefty profit.
Now it seems they weren't alone. The Department of Health says a "handful" of other practices in Rhode Island have admitted to doing the very same thing. DOH is not releasing their names until it completes a thorough investigation.
Is this just common practice to make a buck? Or some sort of coordinated effort? We don't know yet.
The health department also hasn't determined how effective these non approved IUDs are. In the meantime, women with the devices should use another form of birth control.


text:
On the one hand I love spending hours in the kitchen preparing meals for the family and then somedays I want to be able to prepare something that requires minimal input. Not much time or inclination to be in the kitchen today and as a result made this Thai noodle salad for Seren, my youngest daughter. She loves stir fry but not too keen on salads so I took a bit of a risk. She was very enthusiastic about the ingredients that I was putting in it as I got her to shell a few fresh peas and broad beans. Most children love spicy foods and this salad is just that and beautifully fragrant as well. Seren ate about half of it and says it was "ok but needs improving". I on the other hand thought it was stunning!
My 'other' children had sausage dinner with onion gravy today. The only element of it that I need to work on or source is the sausages. At best a good pork sausage is hard to beat if made with quality ingredients and at worst they represent all that is wrong with the food industry. We are working our way from the latter to the former and I will return to the matter of sausages another time.
Thai Noodle Salad
Thai food is characterised by vibrant colours, a variety of textures and flavours that are hot, salty, sour and sweet and my opinion is one of the great cuisines of the world. This salad encapsulates all of those senses. Do not restrict yourself to the vegetables i've used - use what you like (mango is good). However, to ensure a Thai flavour use the dressing ingredients I've listed and also the herbs to give a beautiful fragrance.
Serves 2, take 3 minutes and costs around £1.20 per portion
200g cold ready cooked rice noodles
1/2 red pepper, finely sliced
handful of shelled peas and broad beans
small piece of cucumber, cut into quaters
small handful of coriander, chopped
3 mint leaves, chopped
1/4 red chilli, chopped
small handful of nuts (dry fry in a pan for 2 mins with a pinch of salt)
1 tbsp soy sauce
2 tbsp fish sauce
juice of 1 lime
1 tbsp caster sugar
few dashes of sesame oil
1) In a large bowl put all the dressing ingredients together and stir to dissolve the sugar
2) Add all the other ingredients and toss together. Leave for 10 minutes for the flavours to mingle. Simple as that!


text:
Our Monopoly board brought us to explore Window Trail which lies inside of Big Bend National Park. This is a moderately difficult trail which covers 3.6 miles in total and covers some beautiful scenery along the way. We were fortunate to be there during the spring when many plants were beginning to bloom and the temperatures were ideal for a hike in the desert. Not to cold and not to warm.
Along this trail we enjoyed a number of great sights including Yucca's and cactus in bloom, a couple of white tail deer, colorful birds, and a friendly road runner. This trail follows the bottom of a canyon most of the way where water drains during heavier rainfalls.
About halfway to the end of the trail we decided it was time to explore a place to take a few minutes to relax and take in the great desert mountain scenery. What's the first thing kids want to do when stopping near rocks? Well climb them of course and this we did as some of the nicest seats were higher up. This is not recommended by the park although we were definitely not the first ones to do this as I found an orange peel in one of the crevices left by a previous climber.
Once entering the canyon portion of the hike there were stairs built to make the trail easier to traverse as during other times of the year there can be water flowing through here which is, of course, the way this canyon was created.
At a couple of points along the way we wondered if we were at the end of the trail. I can say definitively now that the end is very obvious because of seeing the window it was named after and it drops off to the desert floor very abruptly. I'm not sure a person would survive a drop from the window.
As you get close to the window the rocks become extremely slippery due to the running water carrying debris which has sanded down the rocks giving them a polished appearance and feel. It was interesting, especially in this dry season, to see how high the water can get in this canyon and how fast it must rush through to wear down the stone underneath.
Carefully climbing near the end there is just a narrow slot with a large drop-off which is all very slippery requiring some care to get to. It would be interesting to see this area from the other side after a heavy rain. There must be an interesting waterfall for a short time.
During our trek back we encountered this road runner that didn't seem to care we were there as it walked right next to us on the trail. Other hikers behind us had the same experience. This particular bird must be use to hikers on this trail and understand prey can be found scurrying as hikers pass by making them easier to find. It gave us the opportunity to all see a road runner finally as each time before the birds would be gone before everyone could see them running across roads in front of the car.


text:
Shop by
In all of the products shown below, the photo goes behind the window opening. Because of this, a small amount of the outer edge of your picture will be covered up. This is necessary to hold the photo in place without the need for tape or glue, and is the same concept as a picture frame mat.
The lighter edges on the photo (below, right) represent the part of the picture not shown through window opening.
* Our 8" x 10" white vertical conventional portrait folders measure:
** Our 8" x 10" white horizontal conventional portrait folders measure:


text:
The ongoing crossfire between French-owned Credit Lyonnais Bank Nederland and Epic Prods.' Eduard Sarlui and Moshe Diamant escalated yesterday when CLBN filed a cross-complaint claiming the pair "cooked" Epic's company books and "looted" the bank-owned entity of more than $ 200 million. That figure includes $ 47.8 million in outstanding loans to the duo's Trans World Entertainment.
The action marks the latest chapter in ongoing lawsuits that began in August after the bank ousted Sarlui and Diamant from their roles as directors and officers of Epic Prods. The duo sued the bank for$ 100 million-plus for breach of contract and various damages.
This countersuit, filed in Los Angeles Superior Court, charges that documents seized last August prove that Sarlui and Diamant committed fraud and embezzlement by using Epic's corporate funds for everything from "private school tuition for their children" to down payments on homes in Palm Springs and Beverly Hills, not to mention a host of luxury car payments, family vacations in Tel Aviv and "chiropractic equipment for (Sarlui's) son-in-law."
"The bank wants to collect a total of about $ 200 million in damages," according to a CL spokesman, although no amount is named in the suit, which targets Sarlui and Diamant personally as well as a group of TWE companies, several additional companies owned or controlled by either Sarlui and Diamant family members, ACSA, Televix, Global Communications and other entities.
Sarlui and Diamant's attorney Brian Lysaght, of O'Neill & Lysaght, said he hadn't seen CL's cross complaint yet, "but it's nothing that's terribly surprising because we had a stipulation that both sides could amend at any time after Jan. 4, and we had amended and filed our counterclaim some time ago. They're just getting around to filing now."
The detailed, 48-page document alleges Sarlui and Diamant "defrauded" the bank "in every conceivable fashion," including:
o Embezzling company funds to support lavish lifestyles or provide gifts to relatives and friends;
o Diverting millions of dollars of assets to companies owned or controlled by former officers;
o Improperly licensing films on below-market terms to firms owned or controlled by the duo;
o Direct misappropriation of film rights and;
o Acquisition of films from the duo's affiliates.


text:
Saturday, February 13, 2010
E03 Challenge
So, masak issued a challenge in the comments of my last post, and I'd like to throw it open to everyone. Just like we both updated E02 for modern Perl 6, we are going to try to independently update E03 and compare results on Friday, February 19th. This one looks significantly tougher and more interesting than the last, so I'm looking forward to see what other people can come up with!
Subscribe to: Post Comments (Atom)


text:
The Bible Passage: Romans 8:18-39
Title: Preserved to Persevere In Grace
The Bible Passage: Romans 8:18-39
The apostle Paul says, "I consider that our present sufferings are not worth comparing with the glory that will be revealed in us" (v18).
As Christians, we do not wander like aimless vagabonds. Rather, we travel as pilgrims with a destination at heart. Our glorious hope of a renewed creation and resurrected bodies in God's presence made the present hardships we face pale in comparison.
In the midst of all these obstacles, God works out His sovereign purposes for the good of those who love him. He has begun redemption in foreknowing, predestinating, calling and justifying us that we may be shaped into Christ-likeness. If God is for us, who can be against us?
If He has already done the hard part of giving His own Son to us all, how could He not do the easier bit of preserving us till the finishing line? What could possibly separate us from the love of God?
"For I am convinced that neither death nor life, neither angels nor demons, neither the present nor the future, nor any powers, neither height nor depth, nor anything else in all creation, will be able to separate us from the love of God that is in Christ Jesus our Lord" (8:37-39)
"Through many dangers, toils and snares, I have already come,
Tis' Grace has brought me safe thus far, And Grace will lead me home."
- From the hymn 'Amazing Grace'


text:
Every moment that you are alive, you are learning new things. The interesting thing is that our brain doesn't seem to like to learn and change. When we encounter new situations, we try to understand them based on information extrapolated from prior experiences. This seems to be because our brain is locked into a physiological mechanism that requires work.
Romans 12:2 says: "Do not conform any longer to the pattern of this world, but be transformed by the renewing of your mind. Then you will be able to test and approve what God's will is—his good, pleasing and perfect will."
This means that we can't just 'change', instead, we have to train our brain. We have to develop new ways of interpreting data. Out of this renewal, comes a new way of being, doing and understanding.