I never trouble to answer anxiety when trying to sleep buy 5 mg buspar with mastercard, but confine myself without further explanation to exhibiting my possession anxiety or adhd buy buspar 5 mg mastercard. Sometimes the children bring cats with them; once they actually brought two lambs anxiety 5 year old buy buspar 10mg cheap. The animals gazed calmly at each other with their animal eyes anxiety 3 months postpartum buy buspar 5mg, and obviously accepted their reciprocal existence as a divine fact anxiety pictures generic 5mg buspar fast delivery. In that there is certainly no extraordinary mark of fidelity anxiety symptoms joins bones order buspar 10mg overnight delivery, but merely the true instinct of an animal which anxietyzone symptoms buspar 5 mg online, though it has countless step-relations in the world anxiety and panic attacks generic buspar 5 mg without a prescription, has perhaps not a single blood relation, and to which consequently the protection it has found with us is sacred. Page 467 Sometimes I cannot help laughing when it sniffs around me and winds itself between my legs and simply will not be parted from me. Once when, as may happen to anyone, I could see no way out of my business problems and all that they involved, and was ready to let everything go, and in this mood was lying in my rocking chair in my room, the beast on my knees, I happened to glance down and saw tears dropping from its huge whiskers. It has the restlessness of both beasts, that of the cat and that of the lamb, diverse as they are. Sometimes it jumps up on the armchair beside me, plants its front legs on my shoulder, and put its muzzle to my ear. It is as if it were saying something to me, and as a matter of fact it turns its head afterwards and gazes in my face to see the impression its communication has made. Perhaps the knife of the butcher would be a release for this animal; but as it is a legacy I must deny it that. So it must wait until the breath voluntarily leaves its body, even though it sometimes gazes at me with a look of human understanding, challenging me to do the thing of which both of us are thinking. The uncertainty of both interpretations allows one to assume with justice that neither is accurate, especially as neither of them provides an intelligent meaning of the word. No one, of course, would occupy himself with such studies if there were not a creature called Odradek. At first glance it looks like a flat star-shaped spool for thread, and indeed it does seem to have thread wound upon it; to be sure, they are only old, broken-off bits of thread, knotted and tangled together, of the most varied sorts and colors. But it is not only a spool, for a small wooden crossbar sticks out of the middle of the star, and another small rod is joined to that at a right angle. By means of this latter rod on one side and one of the points of the star on the other, the whole thing can stand upright as if on two legs. One is tempted to believe that the creature once had some sort of intelligible shape and is now only a broken-down remnant. Yet this does not seem to be the case; at least there is no sign of it; nowhere is there an unfinished or unbroken surface to suggest anything of the kind; the whole thing looks senseless enough, but in its own way perfectly finished. In any case, closer scrutiny is impossible, since Odradek is extraordinarily nimble and can never be laid hold of. Page 469 He lurks by turns in the garret, the stairway, the lobbies, the entrance hall. Often for months on end he is not to be seen; then he has presumably moved into other houses; but he always comes faithfully back to our house again. Many a time when you go out of the door and he happens just to be leaning directly beneath you against the banisters you feel inclined to speak to him. Of course, you put no difficult questions to him, you treat him - he is so diminutive that you cannot help it - rather like a child. Even these answers are not always forthcoming; often he stays mute for a long time, as wooden as his appearance. Anything that dies has had some kind of aim in life, s6me kind of activity, which has worn out; but that does not apply to Odradek. He does no harm to anyone that one can see; but the idea that he is likely to survive me I find almost painful. This time he covers the distance, without paying any particular attention to the fact, practically in an instant. He is almost at the top, when he stumbles, twists a sinew, and almost fainting with the pain, incapable even of uttering a cry, only able to moan faintly in the darkness, he hears B. A free man, Sancho Panza philosophically followed Don Quixote on his crusades, perhaps out of a sense of responsibility, and had of them a great and edifying entertainment to the end of his days. Naturally any and every traveler before him could have done the same, except those whom the Sirens allured even from a great distance; but it was known to all the world that such things were of no help whatever. The song of the Sirens could pierce through everything, and the longing of those they seduced would have broken far stronger bonds than chains and masts. He trusted absolutely to his handful of wax and his fathom of chain, and in innocent elation over his little stratagem sailed out to meet the Sirens. Now the Sirens have a still more fatal weapon than their song, namely their silence. And though admittedly such a thing has never happened, still it is conceivable that someone might possibly have escaped from their singing; but from their silence certainly never. And when Ulysses approached them the potent songstresses actually did not sing, whether because they thought that this enemy could be vanquished only by their silence, or because the look of bliss on the face of Ulysses, who was thinking of nothing but his wax and his chains, made them forget their singing. But Ulysses, if one may so express it, did not hear their silence; he thought they were singing and that he alone did not hear them. For a fleeting moment he saw their throats rising and falling, their breasts lifting, their eyes filled with tears, their lips halfparted, but believed that these were accompaniments to the airs which died unheard around him. Soon, however, all this faded from his sight as he fixed his gaze on the distance, the Sirens literally vanished before his resolution, and at the very moment when they were nearest to him he knew of them no longer. But they - lovelier than ever - stretched their necks and turned, let their awesome hair flutter free in the wind, and freely stretched their claws on the rocks. If the Sirens had possessed consciousness they would have been annihilated at that moment. But they remained as they had been; all that had happened was that Ulysses had escaped them. Ulysses, it is said, was so full of guile, was such a fox, that not even the goddess of fate could pierce his armor. Perhaps he had really noticed, although here the human understanding is beyond its depths, that the Sirens were silent, and held up to them and to the gods the aforementioned pretense merely as a sort of shield. According to the second Prometheus, goaded by the pain of the tearing beaks, pressed himself deeper and deeper into the rock until he became one with it. According to the third his treachery was forgotten in the course of thousands of years, forgotten by the gods, the eagles, forgotten by himself. As it came out of a substratum of truth it had in turn to end in the inexplicable. In fact, the general opinion at that time was that one simply could not build too slowly; a very little insistence on this would have sufficed to make one hesitate to lay the foundations at all. People argued in this way: the essential thing in the whole business is the idea of building a tower that will reach to heaven. The idea, once seized in its magnitude, can never vanish Page 476 again; so long as there are men on the earth there will be also the irresistible desire to complete the building. That being so, however, one need have no anxiety about the future; on the contrary, human knowledge is increasing, the art of building has made progress and will make further progress, a piece of work which takes us a year may perhaps be done in half the time in another hundred years, and better done, too, more enduringly. There would be some sense in doing that only if it were likely that the tower could be completed in one generation. It is far more likely that the next generation with their perfected knowledge will find the work of their predecessors bad, and tear down what has been built so as to begin anew. Every nationality wanted the finest quarter for itself, and this gave rise to disputes, which developed into bloody conflicts. These conflicts never came to an end; to the leaders they were a new proof that, in the absence of the necessary unity, the building of the tower must be done very slowly, or indeed preferably postponed until universal peace was declared. But the time was spent not only in conflict; the town was embellished in the intervals, and this unfortunately enough evoked fresh envy and fresh conflict. In this fashion the age of the first generation went past, but none of the succeeding ones showed any difference; except that technical skill increased and with it occasion for conflict. To this must be added that the second or third generation had already recognized the senselessness of building a heaven-reaching tower; but by that time everybody was too deeply involved to leave the city. All the legends and songs that came to birth in that city are filled with longing for a prophesied day when the city would be destroyed by five successive blows from a gigantic fist. He could have had as many assistants as he wanted, and indeed he had quite a number, but since he took his job very seriously he insisted on going through all the accounts again himself, and so his assistants were of little help to him. It cannot be said that he enjoyed the work; he carried it out simply because it was assigned to him; indeed he had frequently applied for what he called more cheerful work, but whenever various suggestions were put to him it turned out that nothing suited him so well as his present employment. Quite apart from the fact that in this case the work involved would not be less, only more petty, the great Poseidon could hold only a superior position. And when he was offered a post unrelated to the waters, the very idea made him feel sick, his divine breath came short and his brazen chest began to heave. As a matter of fact, no one took his troubles very seriously; when a mighty man complains one must pretend to yield, however hopeless the case may seem. No one ever really considered relieving Page 478 Poseidon of his position; he had been destined to be God of the Seas since time immemorial, and that was how it had to remain. What annoyed him most - and this was the chief cause of discontent with his job - was to learn of the rumors that were circulating about him; for instance, that he was constantly cruising through the waves with his trident. As a result he had hardly seen the oceans, save fleetingly during his hasty ascent to Olympus, and had never really sailed upon them. He used to say that he was postponing this until the end of the world, for then there might come a quiet moment when, just before the end and having gone through the last account, he could still make a quick little tour. People began to notice us, they pointed at us and said: Those five just came out of that house. It is also pointless for the five of us, but here we are together and will remain together; a new combination, however, we do not want, just because of our experiences. Long explanations would almost amount to accepting him in our circle, so we prefer not to explain and not to accept him. No matter how he pouts his lips we push him away with our elbows, but however much we push him away, back he comes. And you are watching, are one of the watchmen, you find the next one by brandishing a burning stick from the brushwood pile beside you. We are convinced that these ancient laws are scrupulously administered; nevertheless it is an extremely painful thing to be ruled by laws that one does not know. I am not thinking of possible discrepancies that may arise in the interpretation of the laws, or of the disadvantages involved when only a few and not the whole people are allowed to have a say in their interpretation. For the laws are very ancient; their interpretation has been the work of centuries, and has itself doubtless acquired the status of law; and though there is still a possible freedom of interpretation left, it has now become very restricted. Moreover the nobles have obviously no cause to be influenced in their interpretation by personal interests inimical to us, for the laws were made to the advantage of the nobles from the very beginning, they themselves stand above the laws, and that seems to be why the laws were entrusted exclusively into their hands. There is a tradition that they exist and that they are a mystery confided to the nobility, but it is not and cannot be more than a mere tradition sanctioned by age, for the essence of a secret code is that it should remain a mystery. Some of us among the people have attentively scrutinized the doings of the nobility since the earliest times and possess records made by our forefathers - records which we have conscientiously continued - and claim to recognize amid the countless number of facts certain main tendencies which permit of this or that historical formulation; but when in accordance with these scrupulously tested and logically ordered conclusions we seek to adjust ourselves somewhat for the present or the future, everything becomes uncertain, and our work seems only an intellectual game, for perhaps these laws that we are trying to unravel do not exist at all. There is a small party who are actually of this opinion and Page 482 who try to show that, if any law exists, it can only be this: the Law is whatever the nobles do. This party see everywhere only the arbitrary acts of the nobility, and reject the popular tradition, which according to them possesses only certain trifling and incidental advantages that do not offset its heavy drawbacks, for it gives the people a false, deceptive, and overconfident security in confronting coming events. This cannot be gainsaid, but the overwhelming majority of our people account for it by the fact that the tradition is far from complete and must be more fully inquired into, that the material available, prodigious as it looks, is still too meager, and that several centuries will have to pass before it becomes really adequate. This view, so comfortless as far as the present is concerned, is lightened only by the belief that a time will eventually come when the tradition and our research into it will jointly reach their conclusion, and as it were gain a breathing space, when everything will have become clear, the law will belong to the people, and the nobility will vanish. This is not maintained in any spirit of hatred against the nobility; not at all, and by no one. We are more inclined to hate ourselves, because we have not yet shown ourselves worthy of being entrusted with the laws. And that is the real reason why the party who believe that there is no law have remained so few - although their doctrine is in certain ways so attractive, for it unequivocally recognizes the nobility and its right to go on existing. Actually one can express the problem only in a sort of paradox: Any party that would repudiate not only all belief in the laws, but the nobility as well, would have the whole people behind it; yet no such party can come into existence, for nobody would dare to repudiate the nobility. A writer once summed the matter up in this way: the sole visible and indubitable law that is imposed upon us is the nobility, and must we ourselves deprive ourselves of that one law Usually at about noon the young nobleman in charge of the conscription appears at the entrance of that part of town where a detachment of soldiers, both infantry and cavalry, has been waiting since dawn. He is a young man, slender, not tall, weak, carelessly dressed, with tired eyes, waves of restlessness continually passing through him like the shivers of a fever. Without looking at anyone he makes a sign with a whip, his sole equipment, whereupon several soldiers join him and he enters the first house. A soldier, who knows personally all the inhabitants in this part of town, reads out the list of the inmates. As a rule they are all present, lined up in the room, their eyes fixed on the nobleman, as though they were soldiers already. In this case no one will dare to utter an excuse, let alone a lie, everyone is silent, all eyes are lowered, the pressure of the command which someone in this house has evaded is almost unbearable, but the silent presence of the nobleman keeps everyone nevertheless in his place. This is why he does not flee, he simply goes into hiding, and on learning that the nobleman is in the house he even leaves his hiding place and creeps to the door of the room where he is promptly caught by the soldiers in search of him. Having inflicted no great pain, he drops the whip, half from exhaustion, half from disgust, whereupon the beaten man has to pick it up and hand it to him. Only then may he join the line with the others; incidentally, it is almost certain that he will not be recruited. But it also happens, and this is more frequent, that a greater number of people appear than are listed. There, for instance, stands an unknown girl, staring at the nobleman; she is from out of town, from the provinces perhaps, the conscription has lured her here. There are many women who cannot resist the temptation of a conscription in another town, conscriptions at home meaning something quite different. And, strangely enough, it is not considered disgraceful for a woman to surrender to this temptation; on the contrary, in the opinion of many, this is something women have to go through, a debt which they pay to their sex. The girl or the woman learns that somewhere, perhaps very far away, at the home of relatives or friends, a conscription is going to take place; she asks her family for permission to undertake the journey, which is granted - it cannot very well be refused - she puts on her best clothes, is gayer than usual, at the same time calm and friendly, no matter what she may be like at other times; and yet behind all the calm and friendliness she is inaccessible, like an utter stranger who is on her way home and can think of nothing else. When the family is preparing for the conscription she is given the best place, which is near the door where she has the best chance of being seen by the nobleman and can best see him. She is honored in this way, however, only until the nobleman enters; thereafter she begins to fade. He looks at her as little as at the others, and even when his eye rests on someone, that person is not aware of being looked at. Should the person not on the list be a man, his only desire is to be conscripted with the others although he does not belong to this house. But this too is utterly out of the question, an outsider of this kind has never been conscripted and nothing of the sort will ever happen. Occasionally I walk over to the tavern where they sell a sour beer, occasionally I have even poured away a glass in disgust, but at other times I drink it. I like sitting there because from behind the closed little window, without the possibility of being discovered, I can see across to the windows of our house. But it is also possible that I am mistaken; someone, without my having asked him, once said so, and the general impression of this house front confirms this. Only rarely are the windows opened, and when this does occur it is done by a servant who may lean against the balustrade to look down for a while. It follows therefore that these are corridors where he cannot be taken by surprise. As a matter of fact I am not personally acquainted with these servants; those who are permanently employed upstairs sleep elsewhere, not in my room. I did not dare look at him closely and was about to turn around in the door and leave. Page 487 the guest, however, called me over, and it turned out that he too was a servant whom I had once seen somewhere before, but without having spoken to him. It had already torn my boots and stockings to shreds, now it was hacking at the feet themselves. Again and again it struck at them, then circled several times restlessly around me, then returned to continue its work. A gentleman passed by, looked on for a while, then asked me why I suffered the vulture. Now I realized that it had understood everything; it took wing, leaned far back to gain impetus, and then, like a javelin thrower, thrust its beak through my mouth, deep into me. Falling back, I was relieved to feel him drowning irretrievably in my blood, which was filling every depth, flooding every shore. I had been standing at the helm in the dark night, a feeble lantern burning over my head, and now this man had come and tried to push me aside. And as I would not yield, he put his foot on my chest and slowly crushed me while I still clung to the hub of the helm, wrenching it around in falling. I soon collected myself, however, ran to the hatchway which gave on to the mess quarters, and cried out: "Men! As soon as the top began to spin the philosopher went in pursuit and tried to catch it. He was not perturbed when the children noisily protested and tried to keep him away from their toy; so long as he could catch the top while it was still spinning, he was happy, but only for a moment; then he threw it to the ground and walked away. For he believed that the understanding of any detail, that of a spinning top, for instance, was sufficient for the understanding of all things. For this reason he did not busy himself with great problems, it seemed to him uneconomical. Once the smallest detail was understood, then everything was understood, which was why he busied himself only with the spinning top. And whenever preparations were being made for the spinning of the top, he hoped that this time it would succeed: as soon as the top began to spin and he was running breathlessly after it, the hope would turn to certainty, but when he held the silly piece of wood in his hand, he felt nauseated. The screaming of the children, which hitherto he had not heard and which now suddenly pierced his ears, chased him away, and he tottered like a top under a clumsy whip. At the beginning it was so big that I was afraid, I kept running and running, and I was glad when at last I saw walls far away to the right and left, but these long walls have narrowed so quickly that I am in the last chamber already, and there in the corner stands the trap that I must run into.
He discusses with counsel how much time they need for response and reply anxiety symptoms mayo clinic discount buspar 5 mg free shipping, and whether they need to exceed the usual page limits anxiety head pressure purchase 10mg buspar. He prefers to have 2-4 weeks between the reply brief and the hearing depending on the volume of the filings anxiety symptoms help cheap buspar 10 mg mastercard. If a party presents statistics or surveys anxiety 7 scoring interpretation 10mg buspar with mastercard, he considers whether the material is challenged by the other side anxiety support groups generic buspar 5 mg with amex, and weighs the arguments on both sides anxiety symptoms dream like state cheap 10mg buspar fast delivery. Generally anxiety symptoms one side of body order buspar 10 mg line, that requires consideration of questions such as: "What are you using this for Special Procedural Requirements the standard Contra Costa page limits are 15/15/10 anxiety 1894 by edvard munch buspar 10 mg overnight delivery. Judge Goode does not discourage ex partes or expedited briefing if there is a demonstrated need. Motions for Summary Summary Adjudication Judgment or Reflecting on many years in the complex department, Judge Goode observes that few motions for summary judgment are granted. Generally, if the moving party cites voluminous facts, competent opposing counsel is usually able to identify a material fact that is genuinely disputed. Judge Goode does not participate in settlement of cases assigned to him for trial except in a very rare case, and then, only upon request of all parties. At the Issue Conference, he scrubs the witness estimates to develop a total time estimate which he provides to the jury. He does not enforce time limits as to any individual witness, but does tell counsel when they are running ahead or behind. Judge Goode does not pre-instruct jurors on relevant substantive law at the outset because Judge Goode usually finds that the parties are unable to settle the jury instructions until later in the trial. Judge Goode does pre-instruct jurors on relevant substantive law before final summations by the attorneys. Judge Goode allows use of electronic aids in the courtroom, including computer projections. Judge Goode has allowed witness examination via Skype upon stipulation of the parties. Contra Costa County no longer has official reporters, so all are private and contracted by the parties. No one has ever requested interim summations by the attorneys throughout the trial. No one has ever requested use of deposition summaries (possibly including short quotes) in lieu of reading deposition transcripts. Judge Goode has suggested to counsel the use of juror notebooks, which may include selected key admitted documents, selected substantive jury instructions, photos of witnesses, timelines, glossaries of key terms, and organizational charts. Karnow A-3 Superior Court of California California County of San Francisco County Department 304 - Judge Curtis E. See San litigation Department Department 304 Staff: Clerk: Danial Lemire Phone: 415. As with anything you want the judge anything the filing courtesy paper to read, on the date of filing provide a courtesy paper copy directly to this Department. The usual ex parfe procedures are used to secure permission for an increase in parte procedures usual briefis limits. If you plan to use this, arrange matters with Court Call at least 2 court days in Court Call. Counsel wishing to argue motions know telephone appearances can be frustrating: advance. Counsel provide of actions as this may be the only complete record of actions taken at the hearing. Often the court will first provide an oral tentative, or outline the apparent issues, or Hearings. Applications must note the number of times a similar application has been filed hnr vice. Applications If there federal court) in this state (state or federal court) in the last two years. If there have been indicate of three or more such appearances, please indicate the percentage of time over the last two years proposed unopposed, format, engaged with California cases. The dark secret is that counsel are far better production parties burdens suited than a judge to know what the parties really need and what the burdens of production suited a judge really are. This is done in a single document, appending so much of other papers (such as relevant transcripts of earlier hearings) as the parties served, and (usually none). The Submission is filed, served, and a courtesy hearing, provided copy provided to this Department. Please omit references to these agreed-upon matters in the final agreement version of the Submission. Summary judgment/adjudication o A central benefit of litigating in a complex department is the flexibility of having complex department benefit litigating o early resolution of key issues via a stipulated bench trial. A few suggestions to ensure the judge is spending his limited time spending suggestions thinking about the merits of your papers, as opposed to the minutiae of myriad pointless minutiae myriad of your thinking objections: objections: o Make only objections which are truly material. Sealing entire redaction: Sealing documents or indeed whole pages is generally not justified. Not citing the page in your memorandum of points and authorities suggests the material is surplusage. Do not provide courtesy copies in envelopes marked to suggest envelopes unredacted version. No trial is set in this Department without also fixing hours about the trial length. July Page 5 Page A-8 Superior Court of California California County of San Francisco Department 304 - Judge Curtis E. Karnow Kamow Curtis Complex Litigation - Class Action Materials Materials Complex mnterials Counsel in class action litigation may find these materials useful. Suggestions for trial plans (often useful in approval checklist provided contested motions for certification) are provided at page 5. Defendants undertake discovery they need to defend against the motion, if any, and then file the underlake Z. Plaintiffs then do the discovery they need, if any, to respond to the Opposition, and then file the Opposition, 3. Provide sufficient detail to allow court to make independent evaluation of the strengths and of allow strenglhs weaknesses of the case. Citations of investigation of documents [such a documents and discovery responses] used to value the settlement. Why the time limit to object or opt-out is reasonable (usually 60 days is needed). All agreements on payment of attorney fees, including fee splitting and whether the client has splitting given written approval. A liability reasonably individual carefully drafted trial management plan may be material to convincing the trial judge that the case can be judge that convincing material management certified, based on a showing that all the issues can be tried within a reasonable time. Plans in support of certification forego vague defendants convince the judge generalities, and usually: usually: (1) address each element of the claims and apparently material affirmative defenses, genuinely element of law; accounting for possible difficulties such as choice of law; (2) identify or at least describe the evidence, such as witnesses, and the number of witness, to be witnesses, of evidence, identiff describe used for each element; demonstrate (3) may include a proposed jury verdict form to demonstrate how the issues are to be decided by decided include proposed the jury; the provide realistic allocation (4) provide realistic time estimates for the trial, based on an allocation per witness or at least estimates for witness type. The description of statistical evidence usually is specific enough to estimate the number of witnesses needed to present a valid sample, and witnesses sample, estimate specific enough sample. Under 15 a this system, if (for example) 3 alternates are to be selected, a jury of 15 is prior selected. Motions which seek to exclude evidence must specify the specific draftingthese motions. The court will use deposition Indicate attrial, factors) these (and other factors) to impose overall time limits at trial, per side or party. Parties are strongly encouraged to introduce as evidence compilations or summaries of voluminous documents. The underlying documents should be designated and made available to opposing parties together with the proposed summary. The parties should consider whether the underlying documents must also be admitted into evidence. Counsel should be prepared to provide proposed jury instructions and wording promptly court. Counsel must promptly meet and special qonfer proposed confer on proposed jury instructions and special verdict forms. The proponent must make the originals or duplicates o-riginals duplicates examination or copying, or both, by other parties at a reasonable time and place. Never original show a witness anything other than the original exhibit, and ensure your witnesses anything exhibits. Counsel must advise the Court prior to Consideration the commencement of trial, or as soon as the issue is apparent, of any legal or commencement evidentiary matters that counsel anticipate may require extended time for matters jury. Sidebar conferences are discouraged, and counsel should request conferences with the Court during discouraged, recesses. Sidebar and in-chambers conferences (including for cause challenges conferences (including recesses. These are discussed with counsel at side bar (where objections may be made) before posed to before posed 16 the witness by the Court. The Court will not delay trial between phases in order to accommodate accommodate party. All discovery issues connected with punitive damages must have been damages jury verdict on liability. Counsel must plan for failure of technology and be ready to technology Counsel proceed immediately without it. Motions which seek to exclude evidence must specify the specific specif,rc draftin! Criminal Trialso" Sharifi, "Techniques for Defense Counsel in Criminal Bench Trials," 28 Am. Counsel are expected to stipulate to the admissibility or at least authenticity of writing record. Avoid introducing more pages than needed: witnesses, typically the cover page and the centrally pertinent pages, only, are needed. Ielevaniy objections save time at trial by blocking pointless questions, a goal not furthered when objecting to furthered atirialby blocking pointless deposition transcripts. The underlying documents should be designated and made available to opposing parties opposing available underlying together with the proposed summary. The proponent may use a summary, chart, or calculation to prove the content of sufirmary, chart, photographs voluminous writings, recordings, or photographs that cannot be conveniently examined in court. Wiss Hon, A-20 Standard Discovery Order Issued at Initial Case Management Conference Initial Order discovery from aparty All deadlines to move to compel discovery from a party pursuant to the Code of Alt suspended Civil Procedure are vacated and suspended until otherwise ordered by the Court. No party may move to compel discovery until the parties have had a discovery Court. At least three court days before the conference, counsel must deliver a conference. Counsel obligations must have completed all meet and confer obligations before scheduling a discovery conference. The Presiding Judge decides whether an action is complex within the complex decides Presiding Rule Rules meaning of California Rules of Court, Rule 3. The Clerk of the complex reasonable Court will then set a Status Conference at which the Presiding Judge shall decide Presiding Conference whether or not the action is complex. For requirements Rules Rules assure compliance with the requirements of California Rules of Court, Rules 2. Regareling information If there is a need to shield some confidential information from the parties, the lf is need Regarding Confidential Information (Double Level of Stipulation and Protective Order Regarding eonfidential lnformation (Double Level Proteetive Order $tipulation protective used. Use of these model stipulated protective orders is recommended recommended but not required. All motions and briefs shall conform with the California Rules of motions conform California Rules Court, especially Rule 3. With seq" the consent of counsel for all parties, telephone conferences on simple interim case telephone conferences consent counset mutually convenient scheduled management matters may be scheduled with the Court for a mutually convenient Court management matters telephone conferences time and date - with the scheduling and logistics of such telephone conferences to scheduling be the responsibility of the requesting party/parties. As to any discovery motions, the parties are relieved of the discovery motions, Discovery. Rule California Rules statutory obligation under California Rules of Court, Rule 3. Pursuant to the Order entered by the Presiding Judge on July 19, 2017, this 19,20t7,this designated, complex action was so designated, and single assigned to Department 2 of this Court of before the Honorable Marie S. One extra copy of any such filing shall be (1) electronically served upon fiting electronically Califomia. I 113, conform briefs pu{poses this indicate on the caption page that this matter is assigned for all purposes to Department 2. Communications to this email address are not part of the Communications arc communications with of official court files - just like a paper letter, they are not "filed" documents - and will be documents a subject retained for at least 30 days and then be subject to deletion (destruction) thereafter. Ex parte applications Ex parte applications in this matter shall heard by Department 2, on Department parties between 2:00 Tuesdays and Thursday between 2:00 p. With the consent of counsel for all parties, telephone conferences parties, telephone conferences on simple interim case management matters may be management matters scheduled with the Court for a mutually convenient time and date - with the scheduling scheduling scheduled with - with requesting telephone conferences logistics and logistics of such telephone conferences to be the responsibility of the requesting party/parties. Accordingly, no interrogatories party may party may propound more than 35 special interrogatories total and no party may authenticity admissions propound more than 35 requests for admissions (other than as to the authenticity of more documents) total, without prior court order after demonstration of need and a showing without documents) that other means of discovery would be less efficient. The discovery letter brief may instead be electronically delivered 4 A-30 to Department 2 via email address compleyivilAsamnateocourtalt. Pursuantto non-California specifically does not require any appendix of non-California authorities, unless specifically appendix particular stated by the Court as to a particular motion. The initial Case Management Conference is set for Wednesday, Conference Wednesday, 13. Counsel for all parties shall meet and California confer on all matters set forth in California Rules of Court Rule 3. Status of Pleadings; Pleadings; Status of Discovery, including the deposition of Plaintiff and the initial Discovery, Status production production of documents by all parties; parties; documents c. Conference, rulings the Court made the following rulings at the Conference, which are set forth herein as the formal order of this Court. Counsel for the parties are aware that this case is double-set for trial, but this days. September 27,20L8 the Pre-Trial Conference is set for Thursday, September 27, 2018 at Conference Conference Court. The cut-off date for filing and serving any merits discovery motions is cut-off April 25, 2018. The cut-off date for filing and serving any expert discovery motions is cut-off June 1, 2018. At the same time as the disclosure, the parties shall give written offers of disclosure, shall 7,2018. Counsel for the parties stipulated on the record to waive the notice and record timing requirements of Code of Civil Procedure Section 437c and stipulated to the Section 437c andstipulated timing 3 -t A-35 following briefing schedule and hearing date: Any motion pursuant to C. Section motion pursuant date: briefing 437c shall be filed and served on or before June 14, 2018. All motions in limine, including any motions to bifurcate, shall be filed shall 11. Counsel 4 A-36 admissibility reach stipulations exhibitsn exhibits, and attempt reach stipulations on foundation and admissibility of all trial exhibits. Conference, power blow-ups, available for viewing their demonstrative evidence, such as posters, blow-ups, and Power viewing their demonstrative itself.
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Joint spacing of the five joint systems varies from less than one meter to several meters papa roach anxiety order buspar 10mg on line. In addition anxiety symptoms eye pressure buspar 10 mg discount, joint densities are higher in thinner black shales than thicker black shales because of differences in total organic content (organic content is higher in thinner beds than thicker beds) anxiety in the morning discount buspar 10 mg with amex. Loewy also states that joint porosity decreases with increasing bed thickness anxiety attack order 5 mg buspar mastercard, and that "overall joint porosities in the shales with the largest joint densities are less than 1%" [see Loewy (1995) "Observations and Experimental Data" pages 13-45 anxiety 100 symptoms buspar 5mg overnight delivery, for a thorough discussion] anxiety symptoms vision cheap buspar 5 mg on line. Little literature exists that discusses the natural fracturing in the Silurian and Ordovician anxiety symptoms signs discount buspar 10 mg without prescription. Natural fractures are present however anxiety 10 months postpartum buy 5mg buspar with mastercard, as seen in road cuts through the Utica Shale (Figure 2. In 1997 the Earth Satellite Corporation (1997) performed a remote sensing and lineament analysis of the Appalachian Basin in New York. Release joints, propagating during basin uplift and regional rifting, formed at depth. On sheets 1 and 2, where the upper Devonian sequence is very near the surface, northeast to eastern fractures dominate. On sheet 3, the wide variation in fracture orientation observed is "indicative of the fact that this sheet contains both the northeastern-most portion (mostly Ordovician section of the Appalachian Basin) and the St. Martin Joints in Ordovician-age rocks are more profoundly influenced by preexisting basement structure, the Taconic Orogeny and related structural grain, and by the Salina Salts, which would have transferred "almost all the stress from subsequent tectonism to the overlying sequences and resulted in a muted effect in the underlying Ordovician Rocks" (Earth Satellite Corporation, 1997). The potential for development of fractured shale reservoirs depends upon the presence of extensive natural fractures, and "current ambient stress in New York suggests that subsurface fracture orientations in an eastnortheast direction will likely be most favorable as the major feeders for hydrocarbons into fractured reservoirs in either the source rocks or conduits to other reservoirs" (The Cadmus Group, 1997). The first two factors are largely dependent on conditions present at the site of deposition, and the third is determined by intensity and duration of post-depositional heating, or load metamorphisim due to maximum depth of burial. This also assumes that the natural gas has remained, to some extent, trapped in the source to become a "reservoir. In the Appalachian Basin, darker zones within the Devonian Shale (higher organic content) are usually more productive that the organic-poor gray zones (Schmoker, 1980). Both tables are averages of multiple data points from individual wells and from multiple wells by Devonian Shale member. Measurements of total organic carbon in the Utica Shale have been reported in literature (Hay, 1989, Hannigan, 1994, Ryder, 1998, Wallace, 1988). Source Hay and Cisne, 1989 / Outcrop in Central New York Hannigan and Mitchell, 1994 / Outcrop, east-central New York Wallace and Roen, 1989 / Subsurface and Outcrop 3. The classification scheme for kerogen evolved initially from the optical maceral analysis of coal. The elemental analysis is based on the quantification of the hydrogen/carbon (H/C) and oxygen/carbon (O/C) ratios from Van Krevelen (1961). A plot of the ratios, called the Van Krevelen diagram, was developed to diagrammatically determine kerogen types and thermal maturation. Both shales with these kerogen assemblages are capable of generating liquids and gases. Published Rock-Eval Data for Marcellus and Utica Shales in New York Plotted on a Modified Van Krevelen Diagram. It also is used to high-grade areas for fractured gas shale reservoir potential and as an indicator for investigation biogenic gas within a shale reservoir system. Thermal maturation of the kerogen has been found to also influence the amount of natural gas that can be adsorbed onto the organic matter in shale. Thermal maturation can be determined by several techniques, including Rock-Eval, vitrinite reflectance, thermal altercation index and conodont alteration index. Multiple techniques should be employed to help determine thermal maturity of a shale. Peters (1986) defined the thermal parameters in which Tmax (maximum temperature) can be used to define the dimensions of the oil window (Peters, 1986). Plotting Tmax and hydrogen index can show the thermal maturation and kerogen type of the samples. Published Rock-Eval data for the Marcellus Shale and the Utica Shale in New York State was plotted using the technique after Peters (Figure 3. The samples were from different depths and ranged from central New York to western New York. Reflectance measurements have been extended to particles of disseminated organic matter occurring in shales and other rocks (kerogen) and have been the most widely used technique for determining maturity of source rock. Typical analysis normally shows a distribution of reflectance corresponding to the various constituents or - 19 - D. Because humic or vitrinite particles are generally used for reference to the coalification scale, the mean random reflectance of vitrinite (Ro) is preferred to other particles. In some cases, there may be several groups of vitrinite particles with different reflectance present. In these situations, it is recommended that only the group with the lowest reflectance should be used. There is a general trend of increasing thermal maturity going from western New York toward central New York. No vitrinite reflectance data has been reported for the Silurian or Ordovician shales. Different types of spore or pollen grains can show different sorption values at low levels of maturation. Martin 4 Natural Gas Resource and Production the information and data available regarding the amount of natural gas resource contained in the organicrich shales in New York is dated and only available for the Devonian-age shale. No information or studies have been done for the Silurian shales or Ordovician shales in New York or the Appalachian Basin. No estimates have been published for the technically recoverable reserves in the Devonian Shale in New York. Natural gas production data was not reported to the state of New York prior to 1983. Even though production data is now reported to the State, many of the shale wells producing today are for home or local use and may not be producing at fullest potential or from a reservoir management perspective. Some of these wells are not metered and estimates for annual production are provided. Two wells are producing from Silurian-age shales and no wells are producing from the Utica Shale (Ordovician-age). Estimates range from a low of 225 Tcf to a high of 2,579 Tcf (National Petroleum Council, 1980, Zielinski, 1982). The estimates vary based on type of shale included in the analysis (black and or gray), reservoir thickness and gas content per cubic foot of reservoir. Estimates for the Devonian Shale resource in New York were calculated in the 1980 National Petroleum Council Study, Table 4. Martin New York ranks third in resource, behind Pennsylvania and West Virginia, accounting for 14. The assessment, described as qualitative, broke up the Appalachian Basin into 19 sub-areas. Parameters used to help define boundaries include: thickness of black shale, total organic carbon, thermal maturity of organic matter, and structural complexity (development of natural fractures). In areas where all of the parameters fall within ranges suitable for generation and accumulation of natural gas, a designation of good is used. In areas where a majority of the parameters fall within favorable ranges and the remaining are close to acceptable standards, a designation of moderate is used. In areas where a majority of the parameters do not fall within acceptable ranges, a designation of low is used. Of these six plays, two are rated as good (Play 3 and 15), two are rated as moderate (Play 6 and 17) and two are rated as low (Play 16 and 19) (Charpentier, 1993). Play Number* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Natural Gas Resource Name North-Central Western Lake Erie Eastern Lake Erie Plateau Ohio Eastern Ohio Western Penn-York Southern Ohio Valley Western Rome Trough Tug Fork Pine Mountain Plateau Virginia Pittsburgh Basin Eastern Rome Trough New River Portage Escarpment Cattaraugus Valley Penn-York Plateau Western Susquehanna Catskill Basin Total (Tcf) Mean 25. The average daily flow rate per well (assuming a full 12 months of production) was 1. Thru 2001* (mmcf) Gas Field Lakeshore Belt Naples Dansville Rushville Bristol Southern Erie County Year 1821 1880 1881 1902 1914 Dunkirk 200 10-100 Discovery year is determined as date of first shale gas production for fields with gas production from other horizons. After drilling a few feet into the shale, he observed water and bubbles of gas escaping from the shale. A friend by the name of William Aron Hart, a gunsmith of the village, visited the mill from time to time and became interested in his friends experiment and its outcome (Orton, 1899). William Hart continued working on the hole that Edward had started under the mill until the "drill" broke, leaving a portion of the bar "steadfast in the shale" and shutting off the gas flow in the well. Still not discourage, he set about looking for the gas veins (fractures) that were well known in the area, which had been seeping gas at the surface for centuries. He located his third attempt near one of the largest natural gas seeps on the banks of Canadaway creek. Around 1830, the flow rate from the well was gauged at 880 cubic feet in 12 hours (1. A second well was drilled nearby by a newly formed gas company called Fredonia Gas Light Company. The well produced about 2 Mcf per day and continued to supply the town with natural gas for illumination. Records indicate reports were made of salt water in the wells as early as 1887 and the need to pump the wells up to 4 times a year and to "never shut them in. During these early years, drilling for natural gas expanded geographically as well as geologically in New York and elsewhere across the nation. The oldest established gas shale field in New York is Naples, located in southern Ontario County. The field was discovered in 1880 and 19 wells ranging in depth from 1,220 feet to 1,400 feet were drilled. Other fields discovered in the 19th century include the Lakeshore Field and the Dansville field. The Lakeshore field includes many of the early wells drilled along the shores of Lake Erie following the discovery made by William A. The Rathbone field was discovered in 1931 and thirty-one wells were drilled in the field. Twenty-four wells were producers, four were dry holes, two were plugged and abandoned due to poor performance, and one well produced oil. The wells were typically 9001,500 feet in depth and targeted the Nunda and Rhinestreet shales of the West Falls Formation. Gas shows in many wells were reported during drilling and testing elsewhere in southern New York in subsequent years, yet only a handful of wells were drilled and completed as shale producers. Most of the wells were completed in the Lower Hamilton Group, including the Marcellus Shale. The wells were usually fracture stimulated which sometimes improved production, but other times killed the well. In 1964 the Decker #2 well was drilled in the southern part of the town of Smithfield and encountered gas in the upper Hamilton. County/ Field Steuben Rathbone Livingston Dansville Yates Rushville Ontario Naples Field Discovery Date 1931 1881 1902 1880 Tested/ Completed Formation Rhinestreet Marcellus Hamilton (Marcellus) Marcellus Hamilton (Marcellus) Cum. Although many of the fields were established with only one well, several continued to produce through 2001. Production data is often not measured and produced volumes vary based on the needs of the user. This data is taken from the Annual New York State Oil, Gas, and Mineral Resources report published by the Division of Mineral Resources of New York State. The Ritter #1019-1 well was drilled in June 1989 and was completed open hole in the Rochester Shale. The Neilson #1146-1 well was drilled in January 1990 and was completed open hole in the Sodus Shale. Casing was set (7" diameter) at 1,161 feet and the well reached a total depth of 1,865 feet. They unsuccessfully tested the Marcellus and Rochester shales in their Wonderview Farms 1-A well drilled in Schuyler County in early 2002 and the well was plugged and abandoned. From 1981 to 2001, cumulative production from the two Silurian gas shale wells was 132. In total there were 15 research wells drilled with different objectives and testing protocols used. Several of the wells were cored and thoroughly analyzed, completed, treated and production tested. Several of the wells produced gas for commercial use and four wells are still producing as of 2001. Onondaga Protocol Drill cuttings - chemical analysis, mineralology, organic content, x-ray diffraction, pressure transient testing. Core Drill cuttings - chemical analysis, mineralology, organic content, x-ray diffraction, pressure transient testing. Chemical analysis, mineralology, organic content, x-ray diffraction, pressure transient testing. Drill cuttings - chemical analysis, mineralology, organic content, x-ray diffraction, pressure transient testing. Core Drill cuttings - chemical analysis, mineralology, Organic content, x-ray diffraction, pressure transient testing. There are other properties that are also important in characterizing the reservoir potential of shale. These properties are covered below and information is provided for the shales in New York where available. Unfortunately, there is very little published data on the reservoir properties of the shales in New York. In several fractured shale gas plays, the composition of the produced natural gas impacts economics and provides evidence of microbiologic and thermogenic processes (Walter 1997 and 2001). Unfortunately, very little gas chemistry and gas and water geochemistry is available from shales in New York. Thus it is difficult to attempt to draw comparisons to other gas shale plays, such as the shallow biogenic Antrim shale play in northern Michigan Basin. In this report, six wells with natural gas production from Devonian Shale were analyzed along with one water well and one natural seep. The data shows methane concentrations of 80-95% and concentrations of ethane and propane from 3% to 15%. No detailed geochemistry is available to investigate the biogenic or thermogenic processes. No gas composition data is available for either the Silurian or Ordovician shales of New York. Finding these natural fracture systems are critical to commercial production of natural gas and is considered one of the primary exploration strategies. Identification and characterization of natural fractures is typically done either at the surface through outcrop studies or in-situ through the use of geophysical logs or core. In addition, indications of natural fractures are often associated with natural gas shows while drilling a well, especially on air or under-balanced. In New York, only a minimum amount of oriented core has been taken in the shale reservoir systems for natural fracture characterization and no formation imaging logs or down-hole cameras results have been published for natural fracture characterization. The greatest number of fractures from the core analysis was in the Rhinestreet shale, which was not completed in this well. Unfortunately, due to completion circumstances and poor well performance, no observations can be made for improved well performance related to the presence of orthogonal natural fractures. No core or subsurface natural fracture descriptions are available for the Silurian or Ordovician shales. These different storage mechanisms affect the amount of gas stored as well as the speed and efficiency of gas production. The process of adsorption is controlled by properties such as the amount of organic carbon present, the thermal maturity of the kerogen, reservoir temperature, pressure, in-situ moisture of the shale and gas composition (Hill, 2002). It should be noted that these correlations are specific to a given play and should be calibrated with measured data for different depths and reservoir pressure gradients. Correlations have been developed in several gas shale plays using the density log to calculate the gas in place in shale with calibrated data (Decker, 1993 and Zuber, 2002). This was first established in the Antrim Shale by the Gas Research Institute, and has been further developed in other fractured shale plays (Zuber, 2002 and S. As with the relationship to gas content, this relationship is play-specific and should be calibrated with measured data as the organic material changes with origin and maturity. However, no gas content data or sorption isotherms have been measured in the Devonian Shales of New York or for the Silurian and Ordovician shales. These are the type of data that should be collected to fully understand the storage and transport properties of natural gas in shale. Adsorbed Methane, scf/ton 200 180 160 140 120 100 80 60 40 20 0 0 200 400 600 800 1000 1200 1400 1600 14. Shale can have significant amounts of porosity and significant amounts of oil and free gas in that porosity. Even in older shales such as the Ohio Shale in the Appalachian Basin or the Antrim Shale in the Michigan Basin, porosity can range up to 15 percent, and free gas can occupy up 50% of the porosity. It is important to be able to assess this potential, primarily from well logs, which should first be validated and calibrated from core analyses. Porosities were measured using the mercury intrusion method as well as determined from density data (Kalyoncu, 1979). A wide range of porosity was reported from the core, from a low of 0% to a high of 18.
In root tissue anxiety symptoms gastro buy buspar 5 mg, the transcript abundance of 5 anxiety symptoms muscle twitching buy buspar 10 mg without a prescription,250 genes was significantly altered anxiety symptoms racing thoughts buy cheap buspar 5mg line. After 5 h of root hypoxia anxiety erectile dysfunction order buspar 5mg visa, only 195 genes showed altered expression anxiety 24 safe 5mg buspar, of which more than 85% were up-regulated anxiety workbook for teens purchase buspar 5 mg fast delivery. The expression of 147 and 144 of these genes was still altered after 24 and 168 h of flooding anxiety management buspar 10mg low cost, respectively anxiety symptoms overthinking buspar 10mg without a prescription. Prolonged duration of flooding caused considerably more genes to show altered expression, with 3,728 and 5,066 genes differing in their expression compared with controls after 24 and 168 h of root hypoxia, respectively. In contrast to the early response to hypoxia, approximately two-thirds of these genes were found to be down-regulated. This is consistent with the gene expression response in another flooding-tolerant species, rice (Oryza sativa), in which 1,364 and 1,770 probe sets showed increased and decreased expression, respectively (Lasanthi-Kudahettige et al. About 70% of all differentially expressed genes were classified into the main clusters 4, 7, 13, and 14; the largest cluster, 14, alone contained 26% of all differentially expressed genes. In contrast, members of clusters 13 and 14 exhibited down-regulated gene expression in roots in response to hypoxia; genes in cluster 14 were less strongly reduced than genes in cluster 13. Metabolites listed in red showed significantly lower concentrations in treated trees than in controls. Blue and green metabolites indicate higher and unchanged concentrations, respectively. The high proportion of genes involved in the regulation of transcription, signaling, and hormone metabolism and the numerous genes with changed expression coding for protein kinases indicate the dynamic adjustment of metabolic processes taking place in the stressed plants. Similar changes have also been reported in other studies on the transcriptome of plants stressed by O2 deficiency (Klok et al. Effects on Carbon Metabolism the switch from mitochondrial respiration to fermentation is likely to strongly affect energy and carbon metabolism. Indeed, some important genes involved in glycolysis were up-regulated in roots due to hypoxia. The transcript abundance of key enzymes of this pathway (Plaxton, 1996), phosphofructokinase (protein identifier 695057) and pyruvate kinase (protein identifier 698714), were significantly increased due to hypoxia. Consistent with this, the lactate concentration in flooded roots was six times higher than in controls after short-term flooding but thereafter decreased to control levels. This pattern was also seen in pyruvic acid concentrations that initially increased by a factor of 3 (5 h of flooding) but then dropped to control levels. Similarly increased pyruvate and lactate abundance was recently observed in anoxia-treated Arabidopsis (Branco-Price et al. In this study, the higher demand for carbohydrates required for glycolysis in hypoxia-treated plants was most probably compensated for by increased phloem transport of Suc from leaves to roots, as suggested by the higher Suc concentrations in the phloem of hypoxically treated plants (2. Carbohydrate (A), ethanol (B), and amino acid (C) concentrations in leaves and roots of poplar affected by root hypoxia. Normoxic and hypoxically grown trees were harvested and metabolite concentrations were determined. Statistically significant differences between treatments per carbohydrate species at P, 0. In the roots, altered transcript levels clearly indicated a switch in Suc degradation from cleavage by invertases (protein identifiers 711782 and 574265) to phosphorolytic degradation by Suc synthases (protein identifier 692288;. Moreover, it may be that the observed up to 10-fold decrease in pyrophosphatase transcript levels (protein identifiers 748006, 704742, 707491, 551881, and 664206) caused decreased abundance and activity of this enzyme, resulting in increased levels of pyrophosphate, the substrate for Suc synthase. The observed switch in Suc degradation, which has also been observed in several other studies at the physiological and molecular levels (Guglielminetti et al. Starch biosynthesis and degradation were also found to be affected by hypoxia. Starch production seemed to be slowed as transcript levels of the starch-branching enzyme (protein identifier 589574) and starch synthase (protein identifier 569276) decreased. In contrast, starch degradation was most probably altered because of an observed increase in b-amylase expression (protein identifier 679498) and decreases in a-amylase (protein identifier 577743) and a-xylosidase (protein identifier 564524) expression at the same time. Consistent with these changes in transcript levels, some carbon metabolite concentrations in leaves and roots were altered during hypoxia. As suggested from accelerated Suc and starch catabolism, Glc, Fru, and Fru-6-P concentrations increased in roots, while starch concentration showed a slight, but not significant, decrease after 1 week of hypoxia. Increased carbohydrate levels in the roots of flooded plants have been previously observed in trees and herbaceous species (Albrecht et al. In contrast, the results of this study suggest that improved sugar supply to hypoxic roots occurs in order to compensate for increased carbohydrate demand under these conditions. Interestingly, reduced carbohydrate concentrations have been reported in roots of hypoxically treated flood-sensitive species (Vu and Yelenosky, 1991), highlighting the importance of sugar delivery to roots in tolerating periods of O2 deficiency. An inhibition of respiration under conditions of reduced O2 supply has been observed in herbaceous plants (Geigenberger, 2003). As this inhibition occurs at O2 concentrations higher than the Km of cytochrome oxidase, it has been proposed that under hypoxic conditions respiration is slowed in order to reduce O2 consumption, thereby avoiding anoxia (Geigenberger, 2003). S4) and microarray approaches (Supplemental Table S1), suggesting (1) that any inhibition of respiration occurs at the protein or enzyme activity level, or (2) as also concluded from work on Arabidopsis (Branco-Price et al. The glyoxylate cycle exerts a gluconeogenic function linking lipid degradation and carbohydrate production (Eastmond et al. In this case, an involvement of the glyoxylate cycle in gluconeogenesis is more likely than an anaplerotic role (Smith, 2002). Through the glyoxylate cycle, excess acetyl-CoA resulting from b-oxidation of fatty acids would be channeled into carbohydrate biosynthesis. Stimulated degradation of fatty acids is indeed suggested by the 5-fold upregulation in expression of acyl-CoA synthetase (protein identifiers 709380 and 709180; Supplemental Table S1), an enzyme involved in this pathway in peroxisomes (Fulda et al. Following microarray analysis, fold changes were determined from three biological replicates for each time point/tissue type. To distinguish between clusters, violet triangles are drawn to the left of clusters and cluster numbers are given. The criteria given in "Materials and Methods" were applied, and only genes with greater than 2-fold changed expression (q, 0. Increases in succinate concentrations in roots of 466 Numerous energy-demanding processes were repressed in hypoxically treated poplar (Supplemental. For example, the transcript abundance of many transporters strongly decreased (Supplemental Table S1). Moreover, the increased concentrations of the hemicellulose components Ara and Gal. Consistently, changes in transcript levels suggested that the biosynthesis of cellulose, hemicellulose, and cell wall proteins was inhibited (Supplemental. Eight genes with homology to Arabidopsis cellulose synthases were found in poplar. In the roots, the expression of cellulose synthases required for secondary cell wall formation was dramatically reduced (up to 44-fold; protein identifier 555650). In contrast, the expression of only one cellulose synthase gene (protein identifier 714760) involved in primary cell wall biosynthesis declined, and the extent of decrease (up to 4. These results may indicate the importance of the primary cell wall for cellular integrity, whereas formation of the secondary cell wall seems to be of minor importance and may be initiated after survival of the stress period. However, cellulose degradation was also affected, as the expression of most root-specific cellulase isozymes was strongly Plant Physiol. After 5 h (left squares), 24 h (middle squares), and 168 h (right squares) of hypoxia, roots were harvested and transcript levels determined by microarray analysis. The log2 values of fold changes of genes involved in carbon metabolism are displayed using the color code indicated. Green arrows indicate gluconeogenesis, and orange arrows indicate the glyoxylate cycle. Supplemental Table S5 summarizes the expression data of the relevant genes of lignin biosynthesis together with other genes of the phenylpropanoid metabolism that were present on the microarray. In the roots, all steps of this pathway, starting from the conversion of chorismate to prephenate (catalyzed by chorismate mutase), to the production of Phe from arogenate (by arogenate dehydratase), to its conversion to cinnamate (by Phe ammonia lyase), seemed to be down-regulated. For example, the transcript levels of root-specific Phe ammonia lyase isozymes decreased by 7. In addition, the expression of the root-specific chalcone synthase, a key enzyme of phenylpropanoid metabolism, was reduced by 8-fold (protein identifier 572875). Moreover, the subsequent modifications of cinnamate leading finally to the formation of lignin and flavonoids were repressed. Effects on Nitrogen Metabolism Nitrogen metabolism was studied in this work by analyzing amino acid concentrations in leaves and Plant Physiol. This is in good agreement with previous observations of reduced nitrogen uptake in poplar (Kreuzwieser et al. Consistent with the assumption of reduced nitrogen uptake into poplar roots, the gene expression of enzymes involved in nitrogen assimilation seemed to be down-regulated. Metabolite profiling revealed that flooding induced rapid changes in the levels of a wide variety of amino acids in the roots. Interestingly, changes in amino acids were frequently associated with dynamic changes in the levels of transcripts encoding enzymes involved in their metabolism. In most cases, transcriptional responses appeared to act as a mechanism of counteracting flooding-induced changes in amino acid levels. For example, rapid increases in Gly, Tyr, and Ser were followed by transcriptional up-regulation of enzymes involved in their biosynthesis and down-regulation of enzymes involved in their degradation (Supplemental Table S2; Supplemental Figure S6). Conversely, a significant decrease in Glu at 24 h was accompanied by transcriptional up-regulation of enzymes involved in its biosynthesis and down-regulation of enzymes involved in its degradation. Interestingly, in the case of Lys, which was significantly increased after 24 h of flooding, this pattern of negative feedback was replaced with an apparent positive feedback mechanism, with the transcript encoding dihydrodipicolinate synthase 2 (protein identifier 730895), the enzyme responsible for the first committed step in Lys biosynthesis, being significantly up-regulated by more than 4-fold after 5 h of flooding, before Lys levels had significantly 468 increased. Moreover, the accumulation of Lys was followed by a down-regulation of two enzymes involved in its degradation. For example, a 7-fold up-regulation of the transcript encoding a reversible mitochondrial Asp aminotransferase (protein identifier 362675) at the 5-h time point was followed by a 6-fold decrease in Asp at the 24-h time point. Interestingly, by the time the decrease in Asp was at its most severe (24 h), transcript levels for the mitochondrial Asp aminotransferase had already decreased to the point that they were no longer statistically significant. At this time, the transcript encoding another Asp aminotransferase (protein identifier 71675) had become significantly down-regulated and transcripts encoding two enzymes of Asp biosynthesis, Asp semialdehyde dehydrogenase (protein identifier 564677) and Asp kinase/homoserine dehydrogenase (protein identifier 92760), had already become significantly up-regulated by approximately 4-fold. Consistent with the notion that these transcriptional responses act to maintain homeostatic levels of Asp by reducing its catabolism and enhancing its biosynthesis, Asp levels were no longer significantly decreased by the 168-h time point. Although it is not clear exactly how these transcriptional responses were mediated, the observations above are consistent with the notion that transcriptional responses of genes involved in amino acid metabolism were largely driven by changes in amino acid levels. Surprisingly, the most dramatic increase in abundance of a metabolite (57-fold) was observed for uric acid in hypoxically treated poplar roots. This nitrogen-containing compound is a well-known transport form of symbiotically fixed N2 in the xylem sap of legumes (Reinbothe and Mothes, 1962) but is also a product of purine degradation (Woo et al. The latter is most likely the source of uric acid in this study, as stresses other than hypoxia that affect the nutrient balance of plants such as sulfur deficiency (Nikiforova et al. It has to be tested in further studies if nitrogen is relocated from roots to the shoot (like uric acid or other nitrogen-containing compounds), as suggested by decreasing protein concentrations in roots but increasing concentrations in leaves of flooded poplar (Kreuzwieser et al. Growth and Physiology of the Trees Repressed nutrient uptake/transport and the inhibition of biosynthetic processes are in accordance with the assumption that plants restrict O2 consumption in order to avoid the occurrence of complete anoxia (Geigenberger, 2003). Although (1) the roots of the hypoxia-treated trees switched from mitochondrial respiration to alcoholic fermentation with an assumed Plant Physiol. Given the metabolic analysis performed in this study, which indicates that the availability of both sugars and amino acids was not considerably reduced. In addition, the leaves of poplar were supplied with reduced carbon in the form of ethanol via the transpiration stream, as suggested from high ethanol concentrations in the xylem sap and ongoing transpiration even after 1 week of root hypoxia. However, as the growth of belowground plant parts was not determined, it cannot be excluded that root growth was limited by reduced energy supply. Possible Flooding Adaptations One aim of this work was to identify mechanisms mediating flooding tolerance by comparing the highly Figure 6. A, Two weeks prior to and after starting the hypoxic treatment, shoot lengths were determined. White symbols and bars, Normoxic controls; gray symbols and bars, hypoxically treated plants. As expected, this study revealed numerous pathways connected to carbon and nitrogen metabolism that are altered by hypoxia. These pathways were affected in a similar way in Arabidopsis, although experimental conditions differed from this poplar study (Klok et al. Adaptations seen in all experiments included increased glycolytic flux, modified Suc degradation, and a reduction in energy-consuming processes such as transport processes and biosyntheses. However, analysis of the hypoxic response in poplar provided insights not apparent in studies performed using Arabidopsis that cannot be explained by the different experimental conditions alone. A large difference between Arabidopsis and poplar (and also rice; Lasanthi-Kudahettige et al. In stress-exposed poplar tissue, over 5,000 genes showed significantly altered expression, whereas under similar conditions, the transcript abundance of only approximately 150 genes was changed in Arabidopsis (Klok et al. Differences in gene expression in Arabidopsis studies can partially be explained by the different O2 concentrations applied (Van Dongen et al. However, this was still less than one-third of the changes observed in hypoxiatreated poplar. The observed changes in transcript abundance of genes involved in cytokinin signaling may also indicate down-regulated wood formation (Demura and Fukuda, 2007). Another difference between Arabidopsis and poplar was the different temporal pattern of transcriptome changes. It is suggested that many of the changes in gene expression observed in flooded poplar are related to an improvement of carbohydrate status and to the maintenance of high carbohydrate concentrations to allow for the formation of energy equivalents. Furthermore, changes in transcript abundance in poplar occurred in an organ-specific manner, revealing that the majority of changes at the transcript level were restricted to the site of hypoxia; thus, no general systemic signal appears to be generated to trigger gene expression changes throughout the plant. In poplar, such adaptations usually occur 2 to 3 weeks after flooding treatment and therefore were not observed in this study. In poplar, this is mediated by the stimulation of glycolysis and a steady supply of sugars for this pathway, on the one hand, and the repression of energy-demanding processes, on the other hand. From the large changes in transcript abundance observed, we conclude that not a single pathway but the sum of numerous well-orchestrated processes in carbon and nitrogen metabolism and morphological adaptation is responsible for the high flood tolerance of poplar. Four-week-old stem explants were transferred from sterile agar cultures into plastic pots (12 3 10 3 10 cm) containing a Plant Physiol. Each tree was supplied with 200 mL of a solution containing 3 g L21 complete fertilizer (Hakaphos Blau; Bayer) every 2 weeks and was watered daily with tap water. Experimental Setup At the beginning of the experiment, the shoots of the trees had a size of 20 to 25 cm. The containers were filled with tap water until the water level exceeded the soil surface by 2 to 3 cm. Expression Analysis Technical Manual, using an Affymetrix GeneChip Hybridization Oven 640, an Affymetrix Fluidics Station 450, and a GeneChip Scanner 3500 7G at the appropriate steps. Transcripts called as "absent" and genes with an average raw signal value below 50 in the untreated sample were filtered out. In this procedure, each hypoxically treated set of root samples was tested against all four control sets (each collected in triplicate before and 5, 24, and 168 h after starting the flooding treatment). The same was applied for leaf samples, in which leaves from flooded trees were tested against the controls taken before treatment and after 168 h of flooding treatment. For estimating q values, p0 (an estimate of the total proportion of true null hypotheses) was determined based on the distribution of 61,413 P values using the smoothing method. A stringent approach was used to select genes differentially expressed in poplar roots and leaves. Dendrograms displaying the "natural" groupings within the data are displayed with different color intensities based on sorted fold change data, and distances (4. Ethanol concentrations were determined enzymatically as described previously (Kreuzwieser et al. Metabolites were extracted and derivatized using a method modified from that of Roessner-Tunali et al. Microarray Analysis Microarray analysis of changes in transcript abundance in poplar leaves and roots was performed using a set of 30 Affymetrix GeneChip Poplar Genome Arrays. Twenty-one and nine arrays were used to analyze root and leaf samples, respectively. The root samples from flooded trees taken 5, 24, and 168 h after starting the flooding treatment (nine arrays) and the root control samples collected before and 5, 24, and 168 h after starting the flooding treatment (12 arrays) were analyzed by the microarray technique. For leaves, the controls collected before flooding and 168 h after starting the treatment (six arrays) and the leaves of the flooded poplar at this time (three arrays) were used for analysis. Supplemental Data the following materials are available in the online version of this article. Overview of pathways with significantly changed transcript abundance in hypoxia-treated poplar roots. Detailed view of pathways involved in cell wall biosynthesis (graphical representation extracted from Supplemental. Differentially expressed genes involved in primary energy metabolism in poplar roots.
