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He glanced nervously about him and saw, to his astonishment, that the auditorium. He opened a tattered paperback copy of the.

Have I hoped to live on in my son? In a personality yet more feeble, flickering, and. What can my son do for me?

Ah, it is so brilliantly clear. I shall be in all those who. Have I ever hated life—pure, strong, relentless.

I have but hated myself because I could not bear it. Mann does not reveal his name in the novel, but forty years later he.

Mann then proceeds to describe how, at the age of twenty—three, he first. He was not only entranced by the. Philip pulled out a pocketwatch, studied it for a moment, and then, with great.

Here concludes my introduction to Schopenhauer. His philosophy has such breadth. Hence I have chosen to pique your curiosity in.

But, as you wish, Julius it is. My answer is that I am merely responding to your. Think about it, Julius, you came to see me with a request for an.

Let me expand upon this matter. First, there was a tone of urgency in your. It was particularly important to you that I meet with you.

No, you wanted something. You mentioned that your health was imperiled, and, in a sixty—five—year—old man,. Hence, I could only assume that you.

My lecture today is my. You made a request for help, and I responded. And I offered that to you only as an appetizer, a sampler of what is to.

There is a great deal that I, as your guide to Schopenhauer, can offer you, and I. I can continue practicing as a clinical philosopher—.

Unlike Schopenhauer, I have neither an independent source of. The lecture is over. Though they were closer, four rows of seats still.

So I was startled by the remarkable coincidence of your. As for mechanics, I suggest meeting weekly and splitting our hour: Philip waited two or three minutes and then: I need more time to think it through.

I take off Wednesday afternoons. Shall we meet in my office? How quickly one moves back into the familiar force field of another person!

Only a little time in his presence and—presto—my. How arrogant, how disdainful he is. How uncaring about others. And yet there is.

And if that excerpt he read about. The idea of rejoining the universal oneness without any persistence of me. And what draws Philip to me?

That crack the other day. Make him a legitimate, kosher therapist? I want to sponsor him? Something new was emerging in Johanna—some vague sense that motherhood.

Her summers at the. Schopenhauer country estate were particularly difficult. Arthur and her servants. Because of his fierce jealousy, Heinrich forbade his wife to.

Danzig, and, shortly before the advancing Prussian troops arrived under the command of. There, in a strange city, Johanna gave birth to her second child, Adele, and.

Heinrich, Johanna, Arthur, Adele—Father, mother, son, daughter—the four bound. To Heinrich, Arthur was a chrysalis destined to emerge as the future head of the.

Heinrich was the traditional Schopenhauer father; he. She was the Schopenhauer family. Dangerously vital, she had to be contained, protected, and restrained.

What did she feel? Her husband and provider, Heinrich,. Was he not part of the trap, the seal to her coffin? A talented woman, Johanna.

And her young daughter? Little noticed by Heinrich, Adele was assigned a minor. Father Schopenhauer, heavy with anxiety and despair, lumbered to his death,.

Schopenhauer warehouse and leaping into the frigid waters of the Hamburg canal. There she became the dear. Arthur Schopenhauer was to grow up into one of the wisest.

And one of the most despairing and life—hating of men, a man who. Could we foresee it, there are times when children might seem like innocent prisoners.

Nevertheless every man desires to reach old age Four steps led from the street to a landing with a blue—tiled fountain.

On the left, a. Julius greeted him at the door, escorted him into the office, and. In exceedingly clear language you claimed that our therapy was a.

Research shows that therapy, in any hands, is unsuccessful for about a third of patients. Your only error was to choose the wrong type of therapy for me and then persist.

But still, to ask for supervision from a. It is not entirely accurate to say I got. You did make two statements that stuck with me and may have played.

For a moment Julius fumed about having to ask for details. Did Philip think he. Could he be that much of a space cadet? Finally, he gave in and.

I remember asking your opinion of my evening and. It jolted me to. Always meant to do a study of that. But for our purposes today tell me,.

And to permit me. Here it is straight: The good therapist wants to alleviate suffering, wants to help people grow.

Therapists need to relate to their patients, whereas you care little about how others feel. Take the two of us.

You tell me that, on the basis of my phone call to you, you made the. Yet never did you utter a word of consolation or.

I constructed and delivered an entire lecture for you. But it was all so oblique, Philip. It made me feel like I was. Better for me, much better, if you had been direct, if you.

Nothing monumental, maybe just some. I would have wanted the tools, the ideas,. An open—ended comment is by far the best choice. In our therapy I remember you once said.

My point still stands: If you debrief patients at the end of therapy about the process, what do. They rarely remember an. Why did you remember me so well and value what happened between us so much.

I believe you might have some deep affection for me, and because. First, I want to caution you against the error of.

You crave and value relationships, and you. The truth is—you and I are fundamentally different. Why not remain with the great world spirits?

My educational expenses have. I need to see only a few clients a week to meet my. I live frugally, I wish to acquire nothing except the freedom to pursue what is.

But since it seems important to. Schopenhauer himself said that bipeds—his. He cautioned, however, about. He liked porcupines—they huddled for warmth but.

He treasured his separateness and depended. And how can you not agree. Your point of view leaves no room for friendships. I have not had a. You may remember I was a solitary.

With your contempt and lack of. Julius rose, shaking his head. He poured a cup of coffee for himself and paced back.

You sure as hell are. Nor, incidentally, are you making your friend and therapist,. You seem very strongly focused,.

Philip, so I will be merciful and cut to the chase. Philip, who had been averting his gaze throughout this discussion, looked directly. You know a great deal.

Your interpersonal skills, sensitivity, and awareness need work—a lot of. But I want to be helpful to you. All right, here it is.

For once, Philip was startled. I tell you that I want to earn a living as a therapist and that to do so I. Those are my conditions.

Seventy dollars for a ninety—minute session. What have we been talking about? My question was imprecise. How will it profit me.

The very idea of such a chorus. I make a point of. So let me give you my spiel. First, my approach is rigorously.

As for my goal in the therapy group, I can be real clear about. I maintain a here—and—now. And I further assume that ultimately they. Second—you strive to reveal yourself and to be honest in expressing your perceptions of.

Third—everything must go on inside the. If there is contact between members outside the group, it must be brought back.

Philip sat silently with eyes closed and his forehead resting on his clasped hands. Can you imagine the ethical dilemma that creates for.

To turn my attention. Julius stood up, took his coffee cup to the sink, shook his head, wondered about. I refer to all. The goddamnedest things you worry about!

Schopenhauer tutorial arrangement is not of great import to me. When the topic first. He had much to say of great value to. In so many ways he anticipated Freud, who borrowed his work wholesale,.

If there is no. Socrates said it most. And I also know that Schopenhauer has served you well in many ways. I look forward to seeing you here for your first meeting next Monday.

When Arthur turned nine, his father decided the time had come to take over the direction. His first step was to deposit him for two years in Le Havre at the.

There, Arthur was to learn French,. Expelled from home, separated from his parents at the age of nine? Yet, later in life, Arthur. Something important happened in Le Havre: Arthur felt nurtured and enjoyed life.

For many years afterward he cherished the memory. His letters to his parents were so full of praise for them that his mother felt compelled to.

Another important event took place during his sojourn in Le Havre. Anthime, the Blesimaire son, was the same. The two boys became close in Le Havre and exchanged a few letters after.

Years later as young men of twenty they met once again and on a few occasions. Then their paths and their interests.

When Anthime responded with an offer to manage his portfolio for a fee, Arthur. By that time he suspected everyone and trusted no.

Arthur and Anthime had one final meeting ten years later—an awkward encounter. Arthur described his old friend as. Though Arthur seemed undemonstrative and said that he never again thought of.

Gottfried, it is apparent that he never truly forgot his dead playmate, nor the shock of his. Arthur had little difficulty interpreting the dream.

At that time he was living in. Berlin in the midst of a cholera epidemic. The dream image of a reunion with Gottfried.

He chose to move to Frankfurt,. Philip arrived fifteen minutes early for his first group therapy meeting wearing the same.

Julius once again began to question his decision to invite Philip into the group. Perhaps he had been imbued with chutzpah from the start, but.

His father, who had had a financial. All That, a horse he owned with Vic Vicello, one of his poker cronies. Vic was an elusive. His dad hated his new job, but it kept the family solvent; not only did it pay well,.

He was unknown in Washington, a free soul. His past three years at P. Gambling had been so much more interesting than. He also ran a small bookie operation, where he offered ten—to—one odds to.

Musial—they rarely won, at best once in twenty to thirty bets. Julius ran with like—minded punks, developed the aura of a tough street fighter in order to intimidate would—be welchers, dumbed himself down in class to remain cool, and cut many a school.

Though punishment was meted out—no evenings. Julius had admired the principal, and falling from his grace was such a wake—up call that.

But it was too little, too late; the best he could do was to. As a consequence of this episode, the latter—day Julius had an exquisite sensitivity.

Happens also in families. Many of his improved patients had a hell of a time. Julius crunched through the fallen sycamore leaves and strode into the front door of.

Roosevelt High, searching for a master strategy to make himself over. The election bid was a long shot, beyond long shot—longer odds than betting on.

Not in a thousand years. But that was the. What was the worst that. His name would be out there, and all would recognize Julius Hertzfeld as.

Of course, his opponents would dismiss him as a bad joke, a gnat, an unknown. Expecting such criticism, Julius readied himself and prepared a riff about.

He had the gift of gab, honed by long hours in the bowling alley of wheedling. The new Julius had nothing to lose and fearlessly.

As things turned out, not only did Julius recreate himself, but he damn near won. With a football team that had lost eighteen straight games and a basketball.

The two other candidates were. Catherine Shumann, the brainy daughter of the diminutive long—faced. Richard Heishman, the handsome, red—haired, red—necked football halfback, had a great.

Julius rode the crest of a robust protest vote. In addition, to his great. They loved him, the love of the timid, hesitant, make—no—waves Mason—Dixon Yid for the.

So much reinforcement did he. The three Jewish high school fraternities vied for him; he was perceived as. Miriam were soon inseparable.

She introduced him to art and aesthetic sensibility; he was. Yes, chutzpah had taken him a long way. He cultivated it, took great pride in it,.

More than once Julius had erred by attempting to do more. So was it compassion or sheer clinical tenacity that led Julius to think he could yet.

Or was it grandiose chutzpah? He truly did not know. As he led Philip to. Julius felt a wave of compassion and, in his softest, most comforting voice, offered.

Philip stopped in the hallway at the door to the office in which they had met a few. The furniture in the room was simple. This is my library and group room.

When I come in at four—thirty, we start pretty. To ease my billing and bookkeeping task, everyone pays at. Philip shook his head no and looked around the room, inhaling deeply.

He remained standing and industriously began. Despite the bustle of their entrance, Philip did not turn his head. Over his thirty—five years of leading groups, Julius had seen a lot of folks enter.

The pattern was predictable: Occasionally, a newly formed group, which. Once in a while newcomers jump right into the discussion, but generally they are.

But a new member so indifferent that he turns his. Never before had Julius seenthat. Surely, Julius thought, he had made a blunder by inviting Philip into the group.

Having to tell the group about his cancer was more than enough on his plate for the day. What was going on with Philip? Was it possible that he was simply overcome by.

It would be a pleasure to sit back and enjoy the blistering group attack that. Julius did not often remember joke punch lines, but one that he had heard years ago.

Yes, he was all grown up. And he was the therapist of the group. And it was his job. Time to get started. Only after Julius vigorously gestured with his eyeballs to one of the empty.

I met with Philip last week, and. Just at that moment Stuart, rushing in from the pediatric clinic at the hospital and.

All members then turned to Philip, and four of them. Great to see you. Glad to have you. We need some new blood—I mean new.

The remaining member, an attractive man with a prematurely bald pate flanked by. Gill turned toward the familiar faces in the group and began.

But her therapist believes. And her therapist swears by it. So, for months, as. No time for anything else.

No other topic of conversation. Our sex life is defunct. She wanted me to. Portland and appeared at the therapy session this morning carrying his beat—up suitcase.

Rose just unloaded on him and kept on unloading. This is the first time I met him. He was amazing—God, I wish I had a father like.

Just sat there and took it and told Rose that, if she had all that anger, it was probably. He just kept gently denying all her crazy charges and took a guess—a.

It was starting to rain, and I. That was about five hours ago. She never did show up at. I drove over to Golden Gate Park and been walking around since.

Julius, not happy with the pressure the group was applying he had seen too many. Yeah, I see what you mean.

I need guidance—this may. Heard from everyone but you, Julius. And of course from. But here it is. Another reason is that everything I know about Rose and.

What I can do is help you focus on how you contribute to your life. The group fell silent. Julius was right; Gill did not like that answer.

Rebecca, who had taken out two barrettes and was flouncing her long black hair. But sometimes from the mouth of newborn.

Tony was a swarthy man with deep acne scars on his cheeks and a. But we unfortunate humans are so haunted by the past and future.

Do you know why we so yearn for the golden. Allow me to make one marginal note: I refer to a Nietzsche. A loud silence rang out in the group.

Julius squirmed in his chair,. Bonnie broke the silence. I know I keep yearning for my childhood, but I never understood it.

She is a sinking ship. My advice to you is to jump overboard and start swimming. You gave an answer. Welcome to the group.

Schopenhauer felt this was the universal human condition—wanting,. Gill listened, mouth gaping, gaze fixed on Philip. All eyes were now on Philip, who continued to have eyes only for the ceiling.

Hell, I know this is true for me and the way I relate to my work—. Cool it, Julius, he said to himself,the group needs you;. He had planned to talk about his diagnosis in the group today.

In a sense his hand. He checked the clock. Not enough time to. Julius resolved that he would absolutely begin the next meeting with the. When the nine—year—old Arthur returned from Le Havre, his father placed him in a private.

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This would create a much sharper rift between Japanese authorities and the U. Rallies to mark his death have been held in several cities, and some have turned violent.

He becomesthe fourth person to hold the post in four years. Concannon agreed to at least bring that notion back to colleagues.

It is unclear whether Justine Greening raised these concerns with President Kagame during her recent, low-profile visit to Kigali.

The shortlist includes architects responding and interacting with a medieval relic, s modernism, and professional peers - but here we have an architect taking on an epic, natural, pre-historic monument.

But to his surprise, with the National League leading in the ninth inning, and Braves closer Mark Wohlers on to close it out, Braves manager Bobby Cox called down to the pen.

We got rid of slavery, women got the right to vote, we got child labor laws and we can solve our economic problems, but only if choose to act rather than wish.

The payments to unnamed brokers ran at 15, pounds per quarter. Now he is courting high-net-worth individuals in the Americas to jointly raise the rest of the cash.

Theearliest it could take action is Oct. The district was won by President Barack Obama in the presidential elections of and Experts say it is a similar situation globally, with salaries often rising percent a year.

More than six of every 10 Americansblamed Republicans for the impasse. The problem with retaining him in by using the franchise tag?

Amnesty said the drone strikes in Pakistan could be war crimes if the USA is not taking proper precautions to prevent civilian casualties when targeting terrorists.

Should it stand by the wayside when thecompany sells new shares, its That not onlyincreases the risk of default, it raises and extends the pausein consumption and investment the budget morass has alreadycaused.

All of this makes it possible that Fitch Ratings, whichput the U. Until our political leaders get serious about funding international family planning — and achieving a stable global population — our world will be less secure than it could be.

But lefty Craig Breslow got him to hit into an inning-ending double play. Glutinous folkloric colour modal tunes, brass bands, accordions is splattered on to the canvas, but there is no calm in the music, no gravity or dignity, and the vocal lines share a fault of many verismo operas - that of persistently masturbating towards a climax of explosive melody that it never achieves.

But it is still startling to see how flows into Japan have dominated investment behaviour. He said GM continued on its plan to improve profitability and cash flow.

It impacts the Southern Hemisphere polar jet stream, the fast flowing air currents that encircle the South Pole. While this hole only appears during the spring months, throughout each subsequent summer the high-speed jet stream swings south toward the pole.

Check out these photos that blend places in New York Ci Ferrets are often used as a mammalian BOE policy makers have this week made a string of speeches seeking to explain the framework.

Is there any value left in guidance? The Funny or Die production president, Mike Farah, hadjoined a White House meeting in July to discuss how to use popculture to persuade young Americans to sign up.

A floating carbonprice, or emissions trading scheme ETS that will be linked tothe European carbon market, will start the following day, a yearearlier than planned.

I told him he can call me anytime he needs anything. Should he have gotten up and said a few words to his teammates?

What a ridiculous way to count votes. Now is this something the shareholders wanted or is it just the committee controlling it?

S,, which has doubled in the last four years, heavily populated by illegal immigrants. They will still be here working off-the-books, and more will come..

Our major goal was to analyze the development of human brain tissue and generate a model system we can use to transfer knowledge from animal models to a human setting.

Do not participate in any way. Challenge all charges and invites to join up. It is our only hope of survival. These simulation results were compared with current satellite data.

He said the Brewers will not raise any prices and are allowing fans to combine vouchers that they accumulate throughout the month.

Smith was decent Wednesday, going 4-for-5 with a sack in team drills but struggled in the 7-on-7 segment, although he was certainly better than the fifth-year veteran.

She said it was built to destroy bulk chemical agents "whereverthey are found," but added that there were no current plans touse the new system in Syria.

The SEC alleges that Tourremisled investors about a synthetic collateralized debtobligation. His record may have looked pedestrian, but Lackey posted a 3.

In high school, I used to make educational videos on YouTube. Once I got to Bryn Mawr, I talked to people in the communications department and was hired to make videos to capture the experiences of past, current and future students.

There would just be humiliation. And yet, humiliation is no smaller when it only happens in your town or on your block. People on the way to the inaugural mass were left stranded while others piled onto buses and into taxis.

So we find out from the eu abit sinister. So our fearless islamic leader,keeps quiet and awaits the hoardes from romanian elite society,[theives murdurers and rapists,look out britain].

Whilse our nhs is run by foreigners,and now greeks spanniards etc steal our jobs,as big business say we are unemployable. When these companys are burnt to the ground in a future revolution?.

They wont be smileing then,as theve shunned their own nationals to deprivation. Our leaders are in it for them,and they are all racists against their own people.

And since then, Allen has devoted his musical talent to lifting up and building up a city. Instead, members are demanding special benefits from the USTA, all because the plan entails taking over a cracked, out-of-the-way public sidewalk.

They need to recognize that this type of official extortion is why business is afraid to come to New York. I never anticipated making this move. But at this stage of my career I wanted to build on what I did last season.

If the driver of the Range Rover would have stopped, none of this would have happened. What possibly could be motivating the non-buyers to write negative reviews?

There typically are various subtargets assigned, such as a certain number of men, women, domestic students, international students and minority students.

In my experience, the pressure was on, every single year, to come in on target, both in terms of class size and makeup. I always have concerns.

Our division is very good. Jerry Reese on the other hand is always positive. The harvesting of vast amounts of data for commercial as well as national security purposes can therefore go on - unless of course the legal system produces a judgement that stops it.

There will be a jury. It will be convening in the upper deck, down in the high-priced field level boxes, and out in the bleachers. Diehard Yankees fans, who actually pay their way into the Stadium, will be on the panel.

Founded in , the organization, originally under a different name, aims to provide protection and information to LGBT citizens of Iran and to help them leave, if they decide to.

China is the second largest sweets market behind the U. Will history repeat itself? Several people were injured, with at least three critically injured.

While just as many adult stepchildren have told me they consider that unfair or disrespectful to their actual parents. Many times, the children themselves come up with the term.

But curiously, no money changes hands. Instead, people are exchanging food. This is a food swap organised by Apples for Eggs, a produce-sharing network set up by Vicky Swift, 42, and Sue Jewitt, My parents had divorced by that point but my mum pulled things together and took any work she could — way below her education — including working as a cleaner in a hotel just to support us.

She basically had to make a choice and she did that for us to survive. In the meantime, real consequences are worsening for those in uniform.

The price of recovery will be steep in dollars but more important in the time required to rebuild, opportunity cost and strategic risk. Some of the prisoners shared their modest quarters with other inmates, he said.

They may reject the message, but at least encourage them to hear it. Perhaps they never heard of Enron? The portfolio is 92 percentoccupied.

As much as the central bank likes to tout its independence, policymakers were clearly caught off guard by the blowback, both in Congress and among the public, to unconventional monetary policy.

The perception that the Fed was acting recklessly, even if erroneous, was relatively widespread, even among some respected voices in the economics community.

How much do you change? But if you change too much, what happens is: You thought you made mistakes before? You make even more mistakes. The damage will be insidious," Clapper said.

Clapper even raised the specter of treason, saying financial stress could make his intelligence officers vulnerable to being bought off by foreign spies.

I was crushed when I heard what happened. Indigenous Fijians now make up small overall majority. The golden tone has been exclusively developed for the model, and will be seen as the hero colour for the M4 although other colours will be available.

The quaint siding is broken and cracked. Looters scattered pictures, stuffed animals, Christmas ornaments, letters, shoes and clothing across the dusty floor, making off with more valuable items, like a refrigerator.

Luminant says it offered Ida money for the home, but she declined. We understand the technical trends, operational challenges and the regulatory requirements in what is a fast moving international market.

Release soluble phosphate Perez et al. In order to confirm the usefulness of this cut-off point proposed by Perez et al.

Figure 4 shows that strains T1C, T1H, T3C, P3E, and V2B have a significantly higher activity to other isolates, a result that was not observed in the plate assay possibly because one or more acids involved in the process did not diffuse into the agar so that there was no presence of a solubilization halo.

Also, the assay by Mehta and Nautiyal contribute to reduce costs and efforts in the search of microorganisms with biofertilizing potential.

Developing native biofertilizers contributes to the management of tropical soils with high absorption and fixation of phosphorus due to their high cation concentrations, usually low pH and clay texture, characteristics that have traditionally been amended with an increased use of che- Afr.

The values obtained are similar to the results of Chen et al. Those activities reported show that our isolates have a great potential and an acceptable activity.

Differences in the reported values by other authors may be due to the measurement techniques employed or to the source of phosphorus evaluated.

Occasionally, the measurements with rock phosphate may overestimate the values of in vitro solubilization because this source of phosphorus is composed of several insoluble as well as soluble forms of phosphorus Zapata and Roy, One of the most valuable achievements of our research is having isolated an actinobacteria with the ability to solubilize both tricalcium phosphate and aluminum phosphate; the latter is a source difficult to solubilize because aluminum phosphate is rapidly absorbed in acid soils.

Its fixation rate per unit area is twice the rate in neutral or calcareous soils. Aluminum is also the leading cause of phosphorus precipitation in acid soils Olsen and Watanabe, ; Holford, In addition, it is known that AlPO4 toxicity varies by the complexity of the soils and that actinobacteria have good resistance to aluminum thus their soil colonization is facilitated in presence of this element Shirokikh et al.

Strains with improved phos-phorus solubilizing activity lowered the pH probably due to the production of organic acids, which is one of the phosphorus releasing mechanisms reported in scientific literature Khan et al.

Soils with these characteristics predominate in the Colombian tropics, hence strain T3A is important because it can release phosphorus from different sources.

All the facts mentioned above, render these strains good candidates for the production of new bio-fertilizers with the ability of colonizing crop rhizospheres in tropical soils.

These strains have similar activity to the positive control strain Streptomyces sp. MCR24, which was previously characterized by Franco-Correa et al.

Prior studies have reported that strain swith good phosphorus solubilizing capacity have also a variety of mechanisms to promote plant growth like the strain Streptomyces sp.

For this reason, further studies with these strains designed to evaluate other plant growth promoting activeties, like nitrogen fixation, degradation of complex carbohydrates, synergistic activities, antagonisms and production of plant growth promoting hormones, can make an important contribution to agriculture.

The authors would like to thank the Dra. Sandra Constantino Chuaire for her contribution on the translation of this paper. Introduction to soil Microbiology.

John wiley and sons. Comparative efficacy of qualitative and quantitative methods for rock phosphate solubilization with phosphate solubilizing rhizobacteria.

Plant growth-promoting rhizobacteria PGPR: Burbano H, Silva F Sociedad Colombiana de la Ciencia del suelo.

Isolation and characterisation of phosphate solubilizing microorganisms from the cold desert habitat of Salix alba Linn.

Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Roles in Genesis and Functions.

Springer Berlin Heidelberg, pp. Promotion of growth of bean Phaseolus vulgaris L. Non-streptomycete actino- Salcedo et al.

Fassbender H, Bornemisza E Evaluation of actinomycete strains for key traits related with plant growth promotion and mycorrhiza helping activities.

Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia.

Goodfellow M, Williams S Evaluation of in vitro solubilization potential of phosphate solubilising Streptomyces isolated from phyllosphere of Heritiera fomes mangrove.

A modified plate assay for screening phosphate solubilizing microorganisms. Screening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines.

Isolation and characterization of rock phosphate solubilizing actinobacteria from a Togolese phosphate mine. Climate change and biodiversity in the tropical Andes.

Differences in the efficiency of various soil phosphate tests for white clover between very acidic and more alkaline soils. Solubilization of hardly-soluble AlPO4 with P solubilizing microorganisms.

Study on diversity of Actinomycetes saltand alkaline environments. Cloning of a Serratia marcescens DNA fragment thatinduces quinoprotein glucose dehydrogenasemediated gluconic acid production inEscherichia coli in the presence of stationary phase Serratia marcescens.

Isolation and molecular characterrization of phosphatesolubilizing Enterobacter and Exiguobacterium species from paddy fields of EasternUttar Pradesh.

Kumar V, Narula N Solubilization of inorganic phosphates and growth emergenceof wheat as affected by Azotobacter chroococcum mutants.

Leyval C, Berthelin J Interactions betweenLaccarialaccata, Agrobacteriumradiobacterand beech roots: Influence on P, K, Mg, and Fe mobilization fromminerals and plant growth.

A study of soil bacteria dissolving certain mineral phosphatefertilizers and related compounds. Field studies on two rock phosphate solubilizing actinomycete isolates asbiofertilizer sources.

Rock phosphate solubilizing Streptosporangium isolates from casts oftropical earthworms. Mehta S, Nautiyal CS Murphy J, Riley JP A modified single solution method for the determination ofphosphate in natural waters.

Biodiversityhotspots for conservation priorities. Effect of PsolubilizingAzotobacter chroococcum on N, P, K uptake in P-responsive wheatgenotypes grown under greenhouse conditions.

An efficient microbiological growth medium for screening phosphatesolubilizing microorganisms. Actinomycetes in the rhizosphere of semidesertsoils of Mongolia.

Phosphate solubilizing microorganisms isolated from rhizosphereof maize cultivated in an oxisol of the Brazilian Cerrado Biome. Olsen S, Watanabe F A method to determine a phosphorus absorption maximum ofsoils as measured by the Langmuir isotherm.

Phosphate solubilization byPenicillium spp. Diversity ofactinomycetes isolated from Challenger Deep sediment 10, m from the Mariana Trench.

Isolation and characterization of mineral phosphate-solubilizing bacterianaturally colonizing a limonitic crust in the south-eastern Venezuelan region.

Mobilization of phosphorus in soil in connection with vital activity ofsome microbial species. Secretion of organic acids by phosphate solubilizing bacteria isolated from Oxisols J.

Soil phosphorus dynamics, acquisition and cycling in crop-pasture fallow systems in low fertility tropical soils: Modelling nutrient management in tropical cropping systems.

Organic acids production andphosphate solubilization by phosphate solubilizing microorganisms PSM under invitro conditions.

Rodriguez H, Fraga R Phosphate solubilizing bacteria and their role in plant growth promotion. Effect of combined Afr. Saharan B, Nehra V Plant Growth Promoting Rhizobacteria: Effect of Al, Co, and Pb ions on growthof Frankia spp.

Simultaneous Psolubilizing and biocontrolactivity of microorganisms: Growth promotion of plants inoculated with phosphate-solubilizingfungi.

Diversity of soil actinomycetes in Yunnan, china. Zapata F, Roy R Accepted 20 January, The aim of the present study was to establish a method of EvaGreen real time fluorescence quantitative polymerase chain reaction qPCR for detecting quickly bovine rotavirus BRV in fecal samples from calves with diarrhea.

VP7 gene was cloned into the pMDT vector. The bovine viral diarrhea virus, bovine coronavirus, porcine epidemic diarrhea virus, bovine mycobacterium tuberculosis and negative control were detected with qPCR.

Plasmids in five fold gradients were detected using qPCR. Only BRV displayed amplification curve; cross-reactivity between BRV and the other viruses or bacteria was not observed.

The amplification curve of pMDT vector plasmid diluted in to gradient was typical S shape. The detection limit of qPCR was 8. It can be applied to clinical diagnosis and epidemiological surveys of BRV.

Group A rotaviruses RVA are known to be important viral diarrheal agents in infants and young animals, including calves. The numbers of the rotavirus-associated mortality are estimated to be , in Tate, ; The numbers of deaths is particularly high in developing countries Abe et al.

Even in developed countries, rotavirus remains an important cause of morbidity. Rotaviruses possess 11 segments of double-stranded ribonucleic acid dsRNA and two outer capsid proteins: VP4 encoded by gene segment 4 and VP7 encoded by gene segment 7, 8 or 9 depending on the strain , both of which are independently responsible for virus neutralization Estes, The neutralization specificity related to VP7 is referred to as the G serotype for glycoprotein , and that associated with VP4 is referred to as the P serotype for protease-sensitive protein Anthony et al.

With regard to bovine, at least nine G serotypes G, , 10, 11 and three P genotypes have been found so far Santos and Hoshino, ; Papp et al. A similar finding was reported by Swiatek et al.

Our study found that G6 and G10 serotypes were 29 VP7 and VP4 proteins elicit the production of neutralizing antibodies, define the antigenic specificities, referred to as G type and P type, respectively, and are the major antigens neutralizing immune responses during rotavirus infections Aminu et al.

Rotavirus VP7 gene is highly conservative at both ends of open reading frame ORF , such it is feasible to detect rotavirus serotypes utilizing the rotavirus specific primers.

VP7 has been shown to be involved in the early interactions with cell-surface molecules, during the rotavirus entry process Lopez and Arias, ; Martha et al.

Moreover, PCR can be used to detect rotavirus VP7 not only stool specimens also cerebrospinal fluids and sera Hiroshi et al.

Currently many methods including polymerase chain reaction, PCR are available for detecting rotavirus, especially VP6 antigen Luan et al.

Unfortunately, PCR assays require sophisticated equipment, which is costly to maintain, and must be performed in specialized laboratories Xie et al.

The real-time quantitative PCR qPCR methods are not only fast and accurate, and also can test against different target sequences. Its specificity is very high Okada and Matsumoto, ; Santos and Hoshino, Its ability to quantitatively and specifically detect genes has been invaluable for both research and diagnostic applications Schweitzer and Kingsmore, It has not been applied in detecting bovine rotavirus in China.

TRIzol a nucleic acid extraction reagent; Invitrogen, Beijing, China was added to the collection tube in advance. The supernatants of 62 fecal samples were subjected to ELISA for detecting the presence of RV with commercially rotavirus detection kits Lanzhou Institute of Biological Products Company, Lanzhou, China and following the protocol of the manufacture instructions.

GQ , specific primers were designed using Primer Premier 5. The length of predicting product is bp. The concentrations of primers , , and nM were evaluated.

The formation of primer-dimers was assessed by melting curve analysis. Thus, only those concentrations of primers that showed dimer-free reactions were used for the final analysis.

Primers were synthesized from Takara Bio, Dalian, China. The cells Suocheng et al. Then, the cells were frozen and thawed 2 to 3 times.

The PCR products were electrophoresed on 1. The expected amplicons were bp sizes. Then, pMDT vectors were sequenced and validated.

The positive plasmids were quantitatively measured using the optimized reaction conditions to establish a standard curve with the logarithm values of template starting copy number as the horizontal axis and Ct values as the vertical axis.

The positive plasmids were used as standards pMD-VP7 , which were serially diluted fold gradient to 4.

RNA was reverse transcribed. Meanwhile, the negative control was detected. Meanwhile, the negative control was set. Sensitivity tests The plasmids were diluted on the serial fold dilutions , , , and gradients and detected with the established qPCR to evaluate the sensitivity of qPCR.

Stability For the intra-assay variability, the plasmids in 5 dilution gradients , , and were detected using qPCR. The test was repeated thrice.

The copy numbers, mean, standard deviation and variation coefficient CV were calculated from the standard curve for each dilution gradients. For inter-assay variability, the plasmids in 5 dilution gradients were detected with the qPCR for three times every three days.

The copy numbers, average, standard deviation and variation coefficient were also calculated as the methods mentioned above. The conditions for qPCR were as follows: The result testified they are group A BRV.

Establishment of the standard curve for qPCR The fold diluted plasmid standards 4. Such the standard curve had been established with the logarithmic values of the starting copies as the horizontal axis X and Ct values as the vertical axis Y.

The optimum reaction conditions were as follows: The dynamics curve of qPCR was acquired Figure 2. The recombinant plasmids pMD-VP7 in five fold gradients from 4.

Ct values were Afr. It can be applied to detect diarrhea sample clinically. Sensitivity test results The plasmids in five dilution gradients were detected with the established qPCR Figure 5.

Ct values were The corresponding copy numbers -3 2 1 0 were 1. Therefore, the detection limit of qPCR was 8.

Amplification of BRV in agarose gel electrophoresis. The predicted bp band was found. Stability test results As shown in Table 1, the variation coefficients CV of intra-assay reproducibility and inter-assay reproducibility were 1.

The dynamics curve of qPCR. Such, the standard curve was established Figure 3. The slope, 2 intercept and correlation coefficient R of the standard curve were Gene amplification efficiency E equalled to 1.

It can be calculated that Ct value equated to The results show only BRV displayed amplification curve, the crossreactivity between BRV and other viruses or bacteria was not observed.

SYBR Green exhibits a very strong fluorescent signal, but it has been shown to inhibit the PCR reaction and has a narrow dynamic range and lower reproducibility than other detection chemistries Gudnason et al.

EvaGreen dye performed better than SYBR Green in general, and high reaction efficiencies can be achieved using the dye. In cases where template sequence tends to vary, dye-based detection helps prevent false negatives that might result from base pair mismatches in a sequence-specific probe binding region Anderson et al.

So far, it has not been reported that qPCR is utilized to detect bovine rotavirus in China. The present study was to develop a higher specificity and sensitivity method for detecting BRV VP7 gene in fecal samples using EvaGreen dye.

The standard curve of qPCR. The slope, intercept and correlation coefficient R2 of the standard curve were Specificity of qPCR assay. BRV displayed amplification curve, the cross-reactions between bovine rotavirus and other viruses: Sensitivity of qPCR assay.

M, bp DNA Marker; , padding and negativie control; , the fold serial plasmid dilutions , , , and gradients. The established qPCR was high specificity, sensitivity and stability.

However, because of inadequate conditions and detection of small samples, the specificity of the experimental methods and result repetition need to be improved in the future.

It could be used for clinical diagnosis and epidemiological survey of bovine rotavirus. Failure to genotype herpes simplex virus by real-time PCR assay and melting curve analysis due to sequence variation within probe binding sites.

Rotaviruses and their replication. Lippincott-Roven Publishers, Philadelphia, Detection of bovine rotavirus by TaqMan based Real-time reverse transcription polymerase chain reaction assay.

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. Comparison of multiple dyes for real-time PCR: Sensitive detection of multiple rotavirus genotypes with a single reverse transcriptionreal-time quantitative PCR assay.

Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: Lopez S, Arias CF.

Curr Top Microbiol Immunol Real-time PCR in virology. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications.

Genotyping of rotavirus of neonatal calves by nested-multiplex PCR in India. Okada N, Matsumoto Y Increased detection of rotavirus using a real time reverse transcription polymerase chain reaction RT-PCR assay in stool specimens fromchildren with diarrhea.

Review of group A rotavirus strains reported in swine and cattle. Pathogenesis of intestinal and system in rotavirus infection.

Santos N, Hoshino Y Schweitzer B, Kingsmore S Combining nucleic acid amplification and detection. Detection and analysis of bovine rotavirus strains circulating in Australian calves during and Research priorities regarding rotavirus vaccine and intussusception: Trends in national rotavirus activity before and after introduction of rotavirus vaccine into the national immunization program in the United States, to Pediatr Infect Dis J.

Northwest University for Nationalities. Genotyping of calves rotavirus in China by reverse transcription polymerase chain reaction.

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus. Rapid and sensitive detection of bovine coronavirus and group a bovine rotavirus from fecal samples by using one-step duplex RT-PCR assay.

Effect of eight fermentation medium, different temperature, pH, incubation time, different carbon and nitrogen sources and different concentration of sodium chloride on production of antimicrobial metabolites were studied.

Antimicrobial activity of the fermentation medium was evaluated by cup plate method by measuring the zone of inhibition.

Modified nutrient medium was found to be good base for fermentation. Glucose and ammonium nitrate were identified as best carbon and nitrogen sources, respectively for growth and production of more antimicrobial compounds.

Similarly, initial production medium pH of 7. Optimization of medium and cultural conditions resulted in better antibacterial and antifungal activity.

The zone of inhibition of ERI against Aspergillus niger was 25 and 20 mm for Curvularia lunata, respectively. It is clear that novel Stretomyces strains ERI-1, ERI-3 and ERI produced extra cellular antimicrobial metabolites effective against pathogenic bacteria and fungi, moreover the medium and cultural conditions for better antimicrobial metabolites production have been optimized.

Streptomyces, antimicrobial activity, nutritional requirements, cultural conditions, optimized media. Secondary metabolites produced by them have a broad spectrum of biological activities such as antibacterial streptomycin, tetracycline, chloramphenicol , Arasu et al.

Secondary metabolite production in microbes is strongly influenced by nutritional factors and growth conditions.

A common strategy, widely applied in industrial screening programs, consists of the application of several growth conditions including variables such as production media, incubation time and other factors to different strains which has been studied Vilella et al.

There is strong evidence that this approach improves the chances of finding the desired metabolites Lauren and Yit-Heng, ; Arasu et al.

Considering that any screening program of microbial natural products can handle a limited number of fermentations, applying many growth conditions to each strain may critically restrict the number of strains and therefore the diversity of biosynthetic pathways included in the screening.

As a compromise between these two requirements, a number of conditions are usually applied to each strain Wildmans, Furthermore, without prior knowledge about the preferred growth conditions for a given microorganism, random assignment of media to strains may generate an inefficient redundancy of metabolites or extracts lacking relevant levels of secondary metabolites.

The production of secondary metabolites is affected by the availability of nutrients Valanarasu et al. In fermentation experiments, the production of antibiotics is increased by the presence of a non preferred carbon source, or by other nutrients Mohd et al.

The source and availability of nitrogen can also influence the production of secondary metabolites Arasu et al. In our screening programme for isolation and identification of actinomycetes from Westen Ghats of Tamil Nadu for antimicrobial metabolite production, we isolated actinomycetes from different parts of the forest soil samples.

Actinomycetes recovered from Kanyakumari showed significant activity against most of the tested bacteria and fungi Arasu et al. This study focused on optimizing nutrition and cultural conditions of Streptomyces strain ERI-1, ERI-3 and ERI26 for the production of antimicrobial metabolites under the shake-flask condition.

Effect of different carbon source and nitrogen source for growth and antimicrobial compound production in fermentation medium were evaluated.

Different concentrations of sugar for production of antimicrobial compound and growth were also studied. Influence of different concentrations of sodium chloride in production medium was studied.

Antibacterial assay Test organism The following test organisms were used for antibacterial studies: MTCC , Xanthomonas sp.

After incubation period the culture was diluted. Fungal strains The following fungi were used for experiments: Cup plate method Antibacterial assays Antibacterial activities were assayed by the agar diffusion method by Arasu et al.

The plates were left for 30 min at room temperature for supernatant diffusion. Negative control was prepared using the respective fermentation medium.

A zone of inhibition was recorded in millimeters and the experiment was repeated twice. Antifungal assays Antifungal assay was done by agar diffusion method.

Petri plates Afr. Effect of production of antimicrobial metabolites on different media. Results were analyzed in triplicates. Five mm diameter well was made using sterile cork borer and filled with 0.

All the twelve tested bacteria and yeast growth was inhibited by strain ERI-1 grown in M-1 and M8 medium. M-2 did not show any activity against K.

ERI-3 cultivated in M-3, M-5 and M-8 inhibited the growth of all the tested bacteria; however M-4 was ideal for ERI RESULTS Selection of fermentation medium Effect of pH on the production of antimicrobial compounds Eight different fermentation media named as M-1 to M-8 were used as the base to determine the optimal nutritional conditions for the production of antimicrobial com- Optimum pH for the production of antimicrobial compounds was recorded by diameter of zone of inhibition Table 2.

Effect of pH of the medium on the growth and production of antimicrobial metabolites. Cell growth in terms of biomass was noted in strain ERI-3 as 0.

Effect of incubation time on growth and production of antimicrobial metabolites. Time 24 48 72 Strain S. At 48 and 72 h, it exhibited maximum cell growth as well as antibacterial activity Tables 3 and 4.

It was not suitable for growth also. Effect of glucose concentration on growth and antimicrobial activity Different concentration of glucose on growth and antibacterial activity was studied Table 6.

Effect of different carbon sources on growth and antimicrobial activity Effect of different carbon source on growth and antimicrobial activity was analyzed.

Optimization of antibacterial compound production was carried out in batch culture. Strains were able to grow in all the tested carbon sources Table 5.

However, ERI-1 and ERI-3 exhibited maximum antimicrobial activity in medium supplemented with glucose as a sole carbon source followed by starch and fructose.

ERI was able to utilize glucose for better growth and revealed maximum antibacterial activity. Effect of different nitrogen source on growth and antimicrobial activity The results of nitrogen source utilization are shown in Table 7.

The higher growth and antibacterial activity were observed in ammonium nitrate as nitrogen source followed by sodium nitrate and potassium nitrate in case of ERI-1 and ERI ERI-3 exhibited maximum growth and antibacterial activity by using sodium nitrate followed by ammonium nitrate.

Effect of incubation temperature on growth and production of antimicrobial metabolites. Effect of different carbon sources on growth and antimicrobial activity against S.

Effect of different glucose concentration on growth and antimicrobial activity against S. Effect of different nitrogen sources on growth and antimicrobial activity against S.

Effect sodium chloride concentration on growth and antimicrobial activity against S. Effect of sodium chloride on growth and antimicrobial activity Effect of different concentration of sodium chloride on growth and antibacterial activity was studied.

Increase in concentration of sodium chloride in the medium influenced the growth and antimicrobial compound production Table 8.

Optimized media on antimicrobial activity Optimized media showed good activity as compared to normal fermentation media.

Effect of fermentation media on antifungal activity Fermentation media were also checked against fungal pathogens. Among all the fungi, A. Eight different fermentation media were used for the selection of best antimicrobial metabolite production.

Medium-4 modified nutrient glucose agar media was chosen as the best base for the production and antimicrobial metabolite production for ERI-1 and ERI ERI-3 cultured in M-8 was the best source for antimicrobial metabolite production.

Our results indicated that MNGA was the good base for antimicrobial metabolite production; however Augustine et al.

Optimizations of fermentation conditions are necessary to improve secondary metabolite formation. Dissolved oxygen tension was identified to influence the productivity Arasu et al.

Antimicrobial activity of optimized fermentation medium. Among the fungus, only Candida albicans was used in the initial antimicrobial screening such as effect of different medium, different temperature, different pH and different incubation time, respectively.

Possibility to enlarge secondary metabolite formation with high oxygen levels is a strong dependency of oxygen with the enzymatic reactions of product formation.

It is well known that environmental signals, including pH shock, can stimulate and promote the biosynthesis of secondary metabolites.

We found that medium with glucose and fructose showed maximum antimicrobial metabolite production and cell growth.

Ammonium nitrate and sodium nitrate were found to be the best nitrogen source for the antimicrobial metabolite production. They influence the growth, as well as the antimicrobial metabolite production of the actinomycetes strains.

Our results indicated that more antimicrobial activity was found when the carbon source is glucose in combination with the nitrogen source ammonium nitrate.

The effects of nitrogen sources on streptolydigin production and distribution of secondary metabolites from Streptomyces lydicus were investigated in shake flask level Liangzhi et al.

When soybean meal was used as the source of nitrogen, three analogues of streptolydigin were detected. Among the nitrogen sources glutamic acid was most favorable for the formation of streptolydigin Liangzhi et al.

However maximum growth and antimicrobial activity was obtained in medium supplemented with glucose followed by fructose. Fermentation medium supplemented with glucose enhanced the growth and antimicrobial metabolite synthesis.

The highest activity was obtained in media containing ammonium nitrate as a nitrogen source, followed by sodium nitrate and potassium nitrate.

The results also indicated an incubation time of 96 h as optimal. Gupte and Kulkarni reported that three independent variables, namely concentration of carbon source glucose , nitrogen source soybean meal and temperature of incubation, were found to be the most important for the production of antimicrobial metabolites Gupte and Kulkarni, In general, Streptomyces sp.

Results of the present work indicated that the selected actinomycetes possess antibacterial and antifungal properties in liquid broth. This explains that the antimicrobial metabolites are extra cellular in nature.

Conditions such as pH, temperature and incubation period were optimized. Best me-dium for growth and antimicrobial metabolite production were selected on the basis of antimicrobial activity.

Modified nutrient medium with glucose as a carbon source was found to be good base for fermentation. Best nitrogen source was selected based on the growth and antimicrobial activity.

Antifeedant, larvicidal and growth inhibitory bioactivities of novel polyketide metabolite isolated from Streptomyces sp.

Antimicrobial activity of Streptomyces spp. In vitro antimicrobial activity of Streptomyces spp. Actinomycetes from Western Ghats of Tamil Nadu with its antimicrobial properties.

Production of a growth dependent metabolite active against dermatophytes by Streptomyces rochei AK Nutritional requirements for the production of pyrazoloisoquinolinone antibiotics by Streptomyces griseocarneus NCIMB From natural products discovery to commercialization: A study of antifungal antibiotic production by Streptomyces chattanoogensis MTCC using full factorial design.

Enhancement of kasugamycin production by pH shock in batch cultures of Streptomyces kasugaensis. Metabolic engineering for the production of natural products.

Rare genera of actinomycetes as potential producers of new antibiotics. Antonie van Leeuwenhoek Nitrogen sources affect streptolydigin production and related secondary metabolites distribution of Streptomyces lydicus.

Streptomyces and related genera. Sharpe, and John Holt, editors. Primary, secondary metabolites, H2O2, malondialdehyde and photosynthetic responses of Orthosiphon stimaneus Benth.

Factors affecting antimicrobial activity of Synechococcus leopoliensis. Antifungal and antifeedant activities of extracellular product of Streptomyces spp.

Inhibitors of farnesylation of ras from a natural products screening program. Potential of tropical microfungi within the pharmaceutical industry.



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