Vishwasrao Paresh

"Routine"

2011-01-24T20:17:00.004Z

Voice that calls me of her Own,
And then, I dnt remember what I had Known.
Eyes glittered and hands so subtle,
Back into those Memories, m lost for Real.
All I care is for ur Lovely smile,
Not that far .. r U, when I close my eyes.
Thoughts are dropping like pebble,
Making ripples in mind, so feeble.
Only one thing on the list,
Wana be close to you, For rest of my life .. a final Wish
Every now and then, I skip what I was thinking,
Dunno why, but .. I keep Missing u the Same way as a "Routine" !!

Sid

Rejuvenating ...

2010-11-01T19:27:00.000Z

Drooling and dropping; while witnessing all the uncertainty except ‘The ONE’.

A slow format of unspoken word; with any discussion is always done at the back of the mind. A stem of satisfaction and paradigm of surety is what a person is always in search of. Counting the hours passing for the event to come; an event for virtual and mental happiness is what we want, for which we can accept any kind of adjustments. Appreciating efforts, polite words, understanding the map of fate, logically defining all the justification are in vain if you cannot find ‘The ONE’. All the junk, non-aspiring, down modulated, shapeless, weird, mocking thoughts come to a standstill, when u find the ONE. The tender hovering of ambushed nurturing skills collected in the past years, for ignoring, fails with ‘The ONE’.

Time changes and it changes many things along with the epitome of bondage .....

Rejuvenating, a new vigor of inspiration which breaches in the heart and soul. A place of new hope generates connecting the inner core. With a shining ray of water and the sweet moistness of sunlight, a small plant of a beautiful feelings arise. A peaceful sleep is rather an additional gift !

Taking wrong the turns while the blissful thought are pouring out; whistling on any of the sight with a song and glitter in the eyes. An indication of a person who has found it’s ‘the ONE’! One can only harbor the feeling though many have tired, in a futile way, to describe it; as so like me.

Then there is a thought, a thought of ‘the ONE’, and an unsure wave which was raging so high in the MIND comes to meet the calm shore.

Ganesha

2010-09-29T19:46:00.001Z

More Often ...

2010-09-10T03:43:00.001Z

More Often, You grow on "Me",
And I love this "Me"instead of the "I" inside Me.

More Often, You revolve in my thoughts,
And "eM" left with, the feel of "You", is only that I have got.

More Often, I keep adoring "You" More,
And then, I carefully watch what is coming in store.

More Often, I hear a clear silence at the shore,
Oh yes, "You" and "Your" smile is what I want for sure.

More Often, life is complicate through all the amidst,
And touch of "Your" simplicity makes it up ... for all of the gist.

More Often, I like the way you see "Me" through,
Wondering if I could do the same for "You".

And then, when I dream of "You" in Deep,
Makes me fall "In" your Arms so Asleep.

More Often, I imagine "Your" picture so serene, standing by My side holding the cup of coffee,
And You resting "Your" head on My shoulder makes me feel so free.

More Often, "You" synchronize with the music so soft,
And with "You" so near, "My" heart pounds with a highest knot.

More Often, I stare with all my concentration yet so sane,
And "You" appear from void space to fill out "My" conscience,
Making "Me" realise what I have gain.

More Often, "WE" go for those rides and do the talk,
And the only part I remember is "Your" lovely eyes in that walk.

More Often, at work, "You" call me to tell,
And that brings a smile on my face, I possible cannot ask anything more.

I wish I could help you believe "Me",
The part which I wana say,"I Miss You", feels so amazing you see.

Facebook

2010-09-08T13:12:00.000Z

Facebook

HAVES and HAVE nots ....

2010-09-07T10:14:00.002Z

Not sure whether this is gonna sale over here .. though recently with change of place and re adjusting the formal conjure, one tends to think over the “NEEDS and WANTS” . Well the Needs are for more of basic amenities and Wants as we all know could clubbed into more of pleasure apostle .

To restate the thinking line, there is an expedition among the thoughts to put forth “its” importance. Say, you get to buy a pair of jogging shoes, though while jogging, you cross the street of liquor shop and you roll into a thought of picking the single malt of your own taste. Now you could definitely decipher the HAVE and HAVE nots. Still there could be more basic necessities to be fulfilled than any of the above materialistic thought. This doesn’t mean that you fail to prioritize your Needs over Wants, it’s just that one reaches out inside to rank the HAVES and HAVE nots with the time and resources.

A decision made at any given point is based on few parameters. With HAVES and HAVE nots, the authentication of precise thoughts are enumerated with different parameters of which TIME is constant, rest vary according to necessities.

This trail of ideas could synchronize your 24 hours of the clock. An hour more to the day could formidable solves few issue ? Though it could puncture the plan, while asking the same question of whether the start of the day little earlier could have sufficed the hour ? Time has got no restriction on its walking steps, cos it just doesn’t stop for anything and anyone. To manufacture the wholeness and fruitfully on one HAVE could retrospectively used on other HAVE not’s. This could also lead to decision, in which one HAVE could enclose a window of other HAVE, this doesn’t mean the later HAVE has plunged voluntarily into HAVE nots.

Similarly, to rule out any possibility, one keeps a track reminder somewhere at back of the mind to fulfill its Wants, being aware that it could be replaced with Needs on scale of time.

Lastly, it is an individualistic decision to press upon either of the terms. And to any Downside you have Up to switch to.

Sarfaroshi ki tamanna ...

2010-08-15T07:35:00.001Z

Sarfaroshi ki tamanna ab hamaare dil mein hain.

Dekhna hai zor kitna baazuay qaatil mein hain

Karta nahin kyun doosra kuch baat cheet,

Dekhta hun main jise voh chup teri mehfil mein hain

Ay shaheed-e-mulk-o-millat main tere oopar nisaar,

Ab teri himmat ka charcha ghair ki mehfil mein hain

Sarfaroshi ki tamanna ab hamaare dil mein hai.

Waqt aanay dey bata denge tujhe ay aasman,

Hum abhi se kya batayen kya hamare dil mein hai

Kheeinch kar layee hai sab ko qatl hone ki ummeed,

Aashiqon ka aaj jamghat koocha-e-qaatil mein hai

Sarfaroshi ki tamanna ab hamaare dil mein hai.

Hai liye hathiyaar dushman taak mein baitha udhar

Aur hum taiyyaar hain seena liye apna idhar

Khoon se khelenge holi gar vatan muskhil mein hai

Sarfaroshi ki tamanna ab hamaare dil mein hai

Haath jin mein ho junoon katt te nahi talvaar se

Sar jo uth jaate hain voh jhukte nahi lalkaar se

Aur bhadkega jo shola-sa humaare dil mein hai

Sarfaroshi ki tamanna ab hamaare dil mein hai

Hum to ghar se nikle hi the baandhkar sar pe qafan

Chaahatein liin bhar liye lo bhar chale hain ye qadam

Zindagi to apni mehmaan maut ki mehfil mein hai

Sarfaroshi ki tamanna ab hamaare dil mein hai

Dil mein tuufaanon ki toli aur nason mein inquilaab

Hosh dushman ke udaa denge humein roko na aa

Duur reh paaye jo humse dam kahaan manzil mein hai

Sarfaroshi ki tamanna ab hamaare dil mein hai

http://www.youtube.com/watch?v=qFEEkM49Z6I

A Day, A song and A Thought with a Memory….

2010-08-05T08:47:00.001Z

As the day begins you plan out the work and try to follow the schedule as the day proceeds. But at times you are struck by a thought and it keeps on revolving or fidgeting inside your brain. It takes you a while for you to realize it. Same is with the songs or memories attached to those songs; once it is stuck in your head it keeps on playing or flashing behind your consciences. More or less you keep on murmuring the song, though it has no intension to disturb anyone nor does that song, thought or image contributes to influence to perturb your routine. Then again, it depends upon the "lameness" and of course each thought or a song for that matter which confiscates you, has its own “nuisance value”.

Some memories are indefinitely tuned to the songs, some have been even immortalized forever, tending to walk along with you along the “memory lane”. A thought is different from an idea, so when we refer to a thought it evidently puts oneself on hold or promotes a prompt, slow “back –to-one” smile. Others around you might find that silly or stupid as they are the virtual recipient of your resultant but not of the thought, which has lead to the act. Few thoughts or memories are bound to shoot up with aroma too!!!

A joint study from the University of Virginia and University of Pennsylvania strongly states, that our brain has perceptive regions. Some neuro-cortical regions of brain that can recognise sense of smell or taste, confines the reason why people have differential preferences of coffee or wine.

Coming back to my point, all these process have a regenerative capacity to replenish your thoughts or memoires.

Group : Hoobastank
Song : The Reason Live

http://www.youtube.com/watch?v=RHESGxB5fJk&feature=related

Chapter 7 : Dreaming out ...

2010-07-02T14:40:00.001Z

“.... No, not now please, we are not having this argument right now, “I left with an anguish. I was hungry and agitated from work.

“Ya ..... " the tone slowed by end of the word, "as usual you must be having some important work, which must have been screwed and of course that must have stressed you. Right?" . It was predictable enough to say that. "And now it’s my duty to understand you and let the day end ... so like .... tell me once and for all , m I a doormat or just a disposable napkin for you,” she had out rated her patience and I could figure out that there was no stopping now.

“M, not getting into a fight right now, it’s pretty late. Lavanya is sleeping upstairs, besides what you are doing at so late ?” I tossed a look at my wrist watch… “…. at 2.30 night, don’t you have to drop her school early morning ??”

She left me that smile, which told me I have missed something really important or in spite of reminders I have lost track of time.

“So now for you everything is work, right !?” she turned away from me, saw me sitting near the dining table and so, brought me a glass of water. “Sid, do we have some space in your life. Please help me to understand you better, this relationship needs it right now. “

I knew it was gona come up some day or the other; I have fudged out of the topic, with no escape statement to throw up. And it indeed needed the attention for long. I cannot be foolish now to dope away, else m gona loose something very dear to me. So, I didn’t make any smart ass statement. I was open with all my ears and wish I could have more than two for receptiveness, same time wondering about the stuff m missing her to fill in.

“Yesterday was Lavanya’s last day of her school and ……..” she looked at me, for me to collect, contemplate, connect and remind myself to …

“ …… parent-teachers meeting, oh God I missed it again, didn't I ?? ” I could not gather the courage to look up, felt so ashamed of myself. “ I .... you know I ... no really I ... I will make it next time … I will try my best.”

“Sid, please … “ she instantly moved a step behind, leading me to stop the expression on my face “you very well know it’s not gonna be possible for you and I don’t expect you to be there” she now came forward, putting her hand through my hair getting me close to her … “ but at least discuss things, I need to know if you acknowledge our existence. M apparently lost without you. I m not able to figure out what has got into you … you so engrossed. You have almost given up talking like an adult. You have been missing out the dinner for now like … what an entire month or so ?? You sneak in at night getting the food, grabbing your sleep on the couch.” She rolled her hand over my back. “Baby you are working way too hard, I can’t see you this way. I need you,"she held me to her chest, looking upstairs at our daughters room "...she needs her father too. You are at work for last five weekends straight. M not gona make you talk on that, but hon ..“She perspired her lung out, it was so warm that I could feel it on my forehead. She kissed me on my head and held me tight this time. “Miss you dear, you and our family means more than anything to me, please come back to me…”

"M sorry ...these days m way too occupied .. " I was about to look up and she closed my lips with her finger.

"M not complaining my dear, I believe in you and you hard work, but you have become non approachable. I hate this wall between you and me. I could hardly see you in the morning these days. You have given up communications. Are we in trouble hon ....??"

"No no ... I would never hide anything from you sweetheart."

"So then why haven't I seen you in bed, you look so disturbed all the time. Not been to any social functions. In fact you have given up on your weekly squash and jog. What has been bothering you so much ? There is this cocoon surrounding you, I hate to break it to you.... its lately killing me to see you over stressed."

"The best I can say is .... m trying to work on a project and the government has got its hands involved now. Those people are not so friendly and if this lands in a wrong hand, m afraid of the consequences."

"So honey, then dont get yourself involved !!" she looked at me with a concern that could gawk her family.

"Hmmm....I really should have thought a little earlier before ... anyways" I was helpless to be precise in my word.

Time ... the only irreversible factor !!

2010-06-01T22:10:00.001Z

Dont know whats wrong with ppl and their given time .. and, I always thought when you decide a time one is suppose to observe and try to be on destination within the given time frame ?? Right ?? ... I slightly doubt .. was that only made for BOOK reading !!

.. I mean for once ..... @ max twice, you could be excused ... else it turns into a habit !!

They expect you to be "in-time" and lavishly excuse themselves by either slipping an excuse of parking or mistakenly consider others to be wisely late as they are doing it ... On top of that you get a kiddish smile of sincerity

One, precisely you dont get to choose the time for meeting, its dedicated on terms.

Second, m astonished to see the expression of antiquity of mildness with no concern, not even a glitch ... aaing ??

Third, they dont bother to ask,the only thing you are flooded (instantly) is with their pre-made, pre-cooked and none sense excuses

... Wonder oh Wonder

LOVING ma stay in Pune .. !! :)

2010-05-29T15:19:00.004Z

After long time ... some one dared to use this one ... feeling good !! :)

With no intension of making the flared skid to upstate ..... I tried to make a perpetual conversation in the parking of INOX ..

" Mam .. could you please move a little ... need to get ma vehicle moving" !!

Some dont understand words of respect or rather cant afford to get one :P !! then u have use those which makes them feel disgusting ...

" Madam aho ... haalva aapli, maala pan mazhi kadhayachi ahe " ! .. After lot of fuming and deserted congenial bleeding look she tilted her Activa ...

My efforts to talk was invain so got down from ma vehicle and and asked if she needed some help to move her first ...

" No thank you, I can do it myself .. " a grudge filled reply.

I looked the other end and mumbled in my mouth " Could have done the easy way .. "

" Ooi mister, look here and talk ... Okya na .. !! " petrifying yet angry reaction.

" Yes mam, now could you please do me a favor and get yourself moving ... I dont wana blame you for me being late ..."

" Have given you enough space ... cant you go through " she was annoyed for no good reason ... she herself knew ...

" If I would have, then could have ... wouldn't have faked a courtesy " .... I looked the other way and said softly " at least not for you " ..

" What ... what did you say ... !! Dont try to act over smart, kaalala na .... haan " she howled at me

" Now why would I do that mam, just move a lil ... let me through, thats all I am saying "

The girl on her Activa and her 2 or 3 other girl friends surrounded the place ... they started jabbing among themselves " Bawalatach disto ahe jaara ... baghna aaga thoda pan dheer nahi ... ashya lokana dhaaklun dila pahije .... Picture pahila aala ahe ka , thamasha ... shyaa useless ahe ... "

" Mam .... could you please speak in English ... and for God sake m requesting you for last time ..."

Again girls got into their psycho twitch .... " Murkha ahe ga , nako tondi laago .. ja mhanava ... bommbalayala koni sangitla hota ikda park karyala ... Shiing tar bagh na parking madhe googal lavun english phadto ahe .. Jaude ga kuthe naadhi laagte aslya tawalyanshi ... "

"Hello ....Englsih ...(I was daunted to promote and hear some more .. if you have done with your private discussion ... now ...please" I got a lil pissed and said it aloud , in mean time my friend entered the parking slot to see what is taking so long ...

" Jastach aarda ordi karto ahe ... kuthun kuthun yetata kon jaane, Pune kharab kartat aapla ... fatichar saala ... jasta nautanki karto ahe ... " Girls and their favorite season ... they love to connect all the non-relative stuff .. I have newha understd whats the pleasure behind that... aso !!

My friend came down and she saw the scene ...she heard them talking aloud in Marathi .. with all heartfelt admirable words !!! She was like what the fuck is going on ...whats the matter ... I told her in English that I just wana come out and the girl has parked behind us so got no other option apart from requesting the girl to give a side .. My friend was like " English madhun nako bolus ... tyana nahi kaalnar, thodi gudhaghyat disti ahe .... " ... (haha ... I love to see the CATS fight ...) !

Aaga Porina uchakawayalaa maaja yetta .. :P !! ... I was having fun .. :)

" Ani tu kasla vichar karto ahes ... thoak bindast tichi gaadi ... " my friend said is with all ... audible reachable ... heard-able to the girls (I was laughing inside .. cos now two girls were gona enter the conversation ... )

"REST is Classified ... " ..... GOTTA see a free Show :D !!!! hahahaha

FINALLY the girls gave the side and shallow looks for .... no needed - no fight - none sense conversation ...and their defeat !!

- Parya

An evening : Odyssey

2010-05-20T04:32:00.001Z

It was preplanned and was on verge to get cancel out yet again .. but I had already canceled it once yesterday and so wanted to make this work.

It was 4 pm and fucking hot and m suppose to call before I checkout from house, so did that sincerely and scooped my kinetic to the meeting place near E-SQUARE ... to my surprise I got away with all signals and jotted the place like 10 mins before. I dropped a call once I reached and a voice ova the phn said she will reach by next 10 to the place...

It was pretty sunny, to be true sun was pouring out, God damn thrust was parching my throat .. in-sided the kinetic to the park-slot and climbed the Mall to space out n stroll a little. I wanted to compliment her abt her lovely smile but thought that would be like hitting straight so left the words unsaid.

Girls on their bike are not different than female pilots on plane, haha !! except pilots at least have a sense of direction with more than 1 passenger in plane , so a sense of responsibility :P !!

She made it within time .. (GoTST - Girls n their own standard time) !! :) ... sedulously men are late n ladies are in charge of creating a havoc !! ;) ... in this case the table rounded ...

With no time we reached MOMO' s , food wasn't so tasty so backed out in two , rather the chitni part was too HOT for me. Shifting to next destination, which was LEVEL-9 . An awesome place to hang out, breezed and more of birds from same folk. ( All feather tethered ) :) !!

She was talking & I was listening, for a moment she too thought that she is boring me. But guess tht wasn't really her fault. I had jus bagged a meeting before I headed to her, with those thoughts still whirling it took me another 8 min to get back. I told myself that m wid a lady n I should at least make an effort to respect her time (I have had Mitis bashing me on this for long ...nyays).

We had a nice time, some juice and chat. She talked with no breaks even for breathe and I heard unsettlingly (now thats an understatement :P !! ... still) from her. Her first reaction was that I look like an uncle (i was disheveled, complete pervert feeling for a min .. :) !! and still managed to say words of assertive expedition !! ) , not the exact compliment one like to hear in first meeting ... :D (But I had been tagged as the name , so it didnt mind me nymore) !! But here too she was not at fault. Looks are deceptive but she is yet to know it ( I suppose so ) ;) !!

.... I had to entertain few incoming calls as they were from outside (with few short talk with "yes n no" and without nypoint listening to continues stream of instruction, as if m an "international luggage transporter" !! haha !! ), with she sitting right in front of me and giving the mystique smile to her clenches and node for the distrustful attitude of mine ...

She actually typed a good bye on her mobile and passed me through her screen. (The caller too had impeccable time, she always does !! :) ... haha)

... Can I be more reckless ? :) ... Two girls, one in front of me and other on phone, with complete different topic and M statued for GOOD WHOLE NO reason :) !!

We further prowled ourself to a common exciting place on the ground floor in the international convention center... CROSSWORD, where she made me buy Chetan' s "TWO state". Now I suppose to get back to her afta I have read the book. I donn have an idea of what that book is , so seriously though if it has lot of Love, Romance n bullshit, then m gona drop n offer this book to someone .

Finally we took separate paths afta she dropped me at Odyssey .

Explainable ..... to be !!

2010-05-20T04:30:00.000Z

" No, we dont have a show of IRON MAN II at 2 .... and m here right now !!" she said, with such an incorrigible affection, that left a long lasting effect to change my if at-all plans made for the day ...

My natural reciprocation was .. " then any other .... English pls ... !!"

" ... there is one chick/flick .. betta option, you cant be dealt wid, aahmm till 5.30, you up for a Hindi ? " she was expecting me to not denny ... and I walked right into that !! ...

Now was I suppose to put away an offer of the movie, with no cash outflow from my own pocket :) !! .. (sometimes offers are not so tempting but to easy out on different norms :P !! they dont hurt either ...)

A run on vehicle, which can dissemble any moment of its speed limit betwn 60 to 80 kmph was an experience to drive from home to E-square .. add (afta parking the bike, a thought cross ma mind ... "chaayala ya gaddicha horn sodun sagla waajat ahe ...aso " !! . And on very day you wana observe the commitment of the time, you gotta enjoy all signals (make you smile at them for whole odd 80 sec)

... And I love ma Pune traffic !!! A person can legitimately on wrong side has nerve to say ... " So what.. though m, ovatake from behind ... " and he/she firmly press the point that, " ...the turning signal was provided " .. (and seriously for a sec I felt screaming on top my voice and into there EAR : "aho mala paathila dole nahi ho " ) !! Aapli Gaadi Viman nahi, tari raasta aaplya baapacha naslya mule ... halu chalawanyas maani nahi :D !!

Newhatheless you pressed against the time ... reaching the HALL , you are offered a security check before entering the parking slot... (atta Kineticcha seat khali dicky nahi ... tari pan ... " Ughada lavkar ... checking karyachya ahe " !!) ... I looked straight into the security person's eyes, he got the message and showed me the way...

A soft killing look with no fondness of my efforts was tranquilized to me .... I just dropped a solo ride of explanation to be late ... and walked into the hall ... I gotta admit, if few Bollywood dumpies cannot make originals, then at least copy the movie to the perfection ... (" tikda pan ... chaamari bomabmba ahet !! :) " ) I tried very hard not to sleep but then the movie was working ova my sleeping hormones ... Just that I didn't snore .. !! :P ... usual predictable epilogue and dialouges (high EMO coefficient...martat !!) I was successful in failing for any appreciable words .. though made up my mind not to slosh sarcastic remarks ...

Though nocturnal night life was resumed .... so to depart with a Maagai Pan n Aanda bhurji with another colony friend at midnight @ Swargate on gaadi !!! And Calcuatta sadha masala @ Three star ...

After reaching home !! the other LiFe in another time zone was inevitable .. yet another hr on Facebook and skype !! Sometimes I wish there could have been time and zone transnational machine.

Chapter 10: Reframed

2010-03-11T14:09:00.005Z

She was entering the house, while I was on my way to office. Ours eyes met for a sec, she felt my eyes toddling and surprised. I smiled warmly but was running out of time, so missing out the reaction on purpose, I looked down and adjusted the belt of my wrist watch, glanced into the long mirror next to the door and stepped out quickly.

“Hey wait up, you gotta min ...” she said. I was heading towards my car, making her feel her absence. She walked me out of house early in the morning. She had just returned from her assignment after 3 months of unconditional exiling and as per her convenience we never discussed our work at home so there had been no room for questions at home.

“Nope, sweetie am already running little late for work, catch-ya later?” I tried to drop the conversation impulsively, rushing intemerately without looking back at her, at the same time trailing through the thoughts in my mind. I had couples of things lined up. Her habit of Ignorance is Bliss was lately turning her into an off.

“Listen I get that, you are pissed at me” she chagrin “showing up like this, so early without notifying you, I was little careless and all ….” she flaunted for a moment, soon continued her spawn, slightly rushing and searching for few words in dismay “um … but then, I will watch it next time … oky?” an expression knowingly left for me to react, she further continued “… but I wish to talk you through this …” she was silent on last part of her sentence, felt like, maybe she was opening up. May be, just that she needs some more time to contemplate on what she had been through lately.

“ … Hmm drop a call, will get to you there … was I being rude here, between don’t miss your doctor’s appointment. He calls me to ask about you” simmering my voice and clearing my throat, keeping that to acknowledge her “and m all here clueless mostly, to know whether I have known you for the all this time.” I said with a tone of concern and sarcasm to rage her.

I walked out through the door and I was about to open the door of car and she walked till me. I was now on the other side of an opened door. Next thing I was getting ready, to place myself in the car. As, I watched her making that walk through the door, her picturesque face, bright eyes, baby checks, dark eyebrows and subtle lips approaching towards me.

“You forgot something,” she glared passionately, came close and kissed softly, was quick a genuine one, all from other side of the door. She kept her hand on top my hand through the half opened wind shield. She loves to kiss me before we depart for our work and a quick, hasty hug. “Don’t bother yourself for dinner, make it home early if you can, m in mood to cook today,” and she smiled vigilantly “…. will take care of that for you.” She used to cook when she is off from assignment. Was that a sign that we gonna spend some quality time?? She was fervid as ever.

“May be you should take some time off for a while” I shrug my shoulders “you know.. ahmm …. that’s just a thought, suit yourself or ……. whatever.”

She smiled back “yap dear, see you in the evening, don’t make it late, they can wait, tell them your girlfriend is back and you gonna cut your hours in office.”

“Sweets, that ain´t happening, beside if I tell that, m sure, either of us will get busted in department, we gotta play by the rules” and she smiled as she knew I would say this.

She was character with an utmost understanding inside and an easy aura from outside.

I roared the engine to my Aston and swiftly headed towards the office, an eye was set on her stand still image. Through the side mirror her image faded along as the wheels gathered some distance. I cautiously reminded myself, that I have meetings come up in next month, so I shouldn’t be foolish romantic and do promises which I may not be able to keep.

Sweeping through the day, hardly concentrating on any of the work, keeping a close check on watch, silently wishing if I could push if just a little. Hoping, that day would to get over sooner than usual. It finally ended at office and cursed back with faster than I calculated.

I let myself in, I was greeted with a warm welcome with an ounce of smile. It made me say inside that the equation has gone wrong somewhere, nevertheless without a word, dropping the bag and accommodating a corner of the couch, loosening up the neck tight tie, relaxing with legs stretched. She fetched me a glass of water nearing her into tenderhearted act and ran her finger through my hair.

“Mmm… m I being a trouble here !" her voice narrowed, " M sorry, I didn’t mean to be that way ", and now it started breaking " am …. just that I had to …. but then … so to … means I couldn’t ...and then … any which way … now that I ... I should have ... can we! Can u? … and so … ya, my mistake …” she came down sobbing and sat beside me, resting her head on shoulder, crawling under my arms.

She was baffled, uprooted and ashamed of laying off the relationship with her own terms. She had realized that gaining my trust back is a hard task now.She laid her hand across the chest and I closed her with my other arms. Took a long breath and held her hand softening …. She kept on weeping and those tears finally ran down the check. I have never understood why a lady would, after being cuddled … cry … oozing the liquid like taking permission to be grievous.

We sat unmoved for a while, she dozed off peacefully on my shoulder in next few; being aware that she has already made the arrangements for supper. It was long we shared that passionate, indulgent and cozy feeling. It was a feeling of unbend; unwind more of an affection. I got up slowly lowering her head on the pillow and got her legs up on the couch covering them with my coat. I moved from couch to floor. Folded my hand, rested my head backwards next to her pillow. Remembering the time we spend together in those initial days, oscillating into memorizes. Reframing a situation back into happiness. Thoughts guiding me to bewilderment of meta-phase. Stretching my legs, crossing them over at the end and rushed feeling of satisfaction, I smiled back to myself. Evening was filling its dark shade and afternoon was on its move. Didn’t intend to wake her up, so got up slowly …. Stood and unbuttoned my shirt. She had her legs folded in her knee compacting on the couch. I got on my knee and kissed on one side of her forehead, felt her tender check with back of my hand.

Readjusting and covering her with coat yet again, I took a leave for a shower.

Reality

2010-02-23T22:19:00.003Z

Too often we don't realize
What we have until it is "Gone"
Too often we wait too late to say
"I'm sorry - I was wrong."

Sometimes it seems we hurt the "Ones"
We hold dearest to our "Hearts"
And we allow foolish "Things"
Trust me, "Sarcasm" tears the lives "Apart".

Far too many times we "Let"
Unimportant things into our "Minds".
And then it's usually too "Late"
To see what made us "Blind".

So be sure that you let people know ...
How much they mean to "you"
Take that time to say the "Words"
Before your time is "Through".

Be sure that you "Appreciate"
Everything you've gotta tell "Them"
And be thankful for the moments, however small it maybe.
In life that Means alot for Some, "Value" it.

- sid

Words

2010-02-10T08:14:00.000Z

Expressing things in the form of "Words",
Some are "Soothing", some act like "Sword".

Juliet saying - " I Love You " melted Romeo´s Heart.
Words of "Anger" drive loving one´s apart.

Words of "Consolation" support the falling one.
Words of "Appreciation" makes impossible things done.

Confession of things makes somebody´s life,
And words of "Hatred" hurt heart like knife.

"Words" can "Blossom" the Mood,
Accelerating you, from Low~High to "Good'

Swings your heart and rings an instant patch of "Smile",
Often getting you close to your beloved one´s in spite of your exile.

Some may "Rhyme" and few many flounder, ..... though!

It carries the strength which can "Ignite",
A fire in all coming generation that so bright.

Thats the magic of Words, one has to Understand.

Thus Small "Words", make difference Big,
They can make you beggar or make you king.

Will act of emotion be ever understood ?

So Can unsaid words speak volume,

- Parya.

"Possessionless"

2009-12-08T23:51:00.005Z

All the things that i've collected
Stones and Shells
Every word in every book
Upon my shelves
Only form a brief description of myself
But they don't define who i am
I don't think anything can, no

If i strip away the non-necessities
All the damage all the mess surrounding me
I don't crave what i have not
I don't need more than i've got
It's just me that i offer up

All i got is my body
And it's naked for you
All i got is this heart
That i'm willing to lose
I know in this life
I give it everything
All i got is this soul and
Its shedding its clothes
Do you see who i am now
Your standing this close
You know in these arms
You're feeling everything

I'm learning what to give
What to protect
To look into the mirror
Though i'm not perfect
It's still a work in progress
Hasn't finished yet
Wanna be transparent
See through
Not gonna hide me from you

All i got is my body
And it's naked for you
All i got is this heart
That i'm willing to lose
I know in this life
I give it everything
All i got is this soul and
Its shedding its clothes
Do you see who i am now
Your standing this close
You know in these arms
You're feeling everything

I'm completely undressed
But i couldn't care less
I'm standing here possessionless
It's the only true test
When the only thing left
Is a love thats possessionless
I don't crave what i have not
i don't need more than i've got
It's just me that i offer up

All i got is my body
And it's naked for you
All i got is this heart
That i'm willing to lose
I know in this life
I give it everything
All i got is this soul and
Its shedding its clothes
Do you see who i am now
Your standing this close
You know in these arms
You're feeling everything

-Delta Goodrem

Image of “You” ...

2009-11-18T09:59:00.006Z

.... Your smile is heavenly,
You touch me tenderly,
Your eyes they speak to me, a thousand summer nights.....
Oh, lovely lady.
You mesmerize me,
You fill my heart and soul with pleasure and delight,
Ah feel so complete with you,
I newha wana let you go.
Wonder that´s love I feel, sneak upon me,
Feel I like belong to you, you belong to me,
Tonite, yeah tonite could be the nite you and I become eternally ...

... M already loving D-Day

2009-10-22T17:46:00.010Z

Phone was ringing vociferously with the tone of “Toss of Feathers – The Corrs” the sound tearing apart the silence and flash light beeping showing the name “MUM-Ma”, and now m all of a sudden rearranging and shifting my dreams into thought “I have to know that I have done something wrong or said something irrational (as usual !) or I have failed to call some1 very important (why do I have to do that?) from family on some occasion which I haven´t even heard of (for example Udakshanti .. watewha ..) or maybe I m forgetting (not hard for me to do that repeatedly …ya so !!) or just a reminder call (possible .. for my reckless behavior)??”

The stage was half asleep, as if it is 04.00 hr is morning and yet “ aaamhhh hummm (so, still not sure how to start the conversation), yes M getting up Mom its just 7 here have mercy" and I was saying sorry before hand or thank you (for no reason), "still two more hrs to get to work”

“Happy Birthday Siddhu”.

Now she is the only one, among the family members who gets that right to say and of course meticulously referred back with response of satisfaction. Not because others don’t know that name but mainly others have coined their own term to fondly beckon me with sort of addiction. My bro louvhes to call me “Bundya” a 24 carat, typical maharashtrian name (means: notorious and abide character) or the other hand, my Mama (moms brother), “Shaahana-Karta”, it has story behind it but ya (split meaning: a stupid but a complete smartass).

“ … huh, Oh thankx mom, wasn’t expecting that so early though.”

“Get up, get dressed and have a great day ahead, kaal ratrichi uttarli nahi ka (hope you weren’t high yesterday)” it ended with a giggle.

“No its weekday, Ma” certainly I had those ideas popping in my head last night but needless to say I was aware that it was one more day for weekend to follow.

This conversation was usual for every Saturday morning wake-up call, it wasn’t Saturday (though it felt like) but the tone and voice quality was nevertheless same. Slightly lower pitched!! (Maybe not in mood of scolding, as I could figure out the decibel in acoustics, were astonishingly matching for beloved one´s).

“Bro-Vahini and Sis had dropped call.” Both being in totally different time zone the calls abruptly ended or didn’t, I seriously have no idea cause I was eyes wide shut during that course of time. So I mostly refrained from telling her any conversation (which I still do not recall) if at all I had made with either of them. I faintly recall Tejas, my sis and Dockya, a long substantial friend from (was it him?? Calling details on cell at least show that record ).So brightly speaking this was my first call of the day.

“Yes dear, bless you with all love and let God bestow you with success and joy”, she was happy to say those words as I was happy to hear them. “Mag aaj kahi special karnar ahes ka (so anything something special for the day), you go good oky !! and be happy always.”

I had a faint idea where the talk was heading to. Knowing that direction, I preferred to take another route to talk her out . “Yes, I will take care”. I could hear father from behind telling her to hurry up “aaga chal tyala usher hoil apan sandhyakaali bolu nivanta. (come on, let him get ready don’t let you keep him, else he might get late for his work, we will talk with him later in evening).”

“Ho mag boluya aapan nivanta, raatri. Tu ghari aala ki phone kar oky? Bhetu skype war. (Oky then talk to you later tonight, ring us up when you come online on skype). Have fun.”

I was now getting back to my senses, still being in bed under the warmth of the douve, floundering about how to cope up with the day. Didn’t felt like showing up in lab ( as if anyone had asked my choice or opinion for that matter). The last thing I could visualize was how the last moment celebration was planned and designed. Rewinding 7 to 7.30 hours, now dragging into past.

“ … Ya thank you, m glad that you remembered between what are you doing so late, it must be 3 .30 in India”I said to Shiny as she was the first caller for fetching me advance birthday wishes.

“Waat divsachi hardhik shubhecha ... Nahi Nahi , I was sleeping, alarm laagaya tha maine (I was sleeping actually had put an alarm)”she proclaimed.

We talked for a while and then she dozed of back into her time zone, mostly sleeping I guess.

Shiva was on his laptop watching highlights of football AC Milan vs Real Madrid, a match which AC Milan won and made him happiest person on the earth. One thing was for sure that he might be loving his girl friend but not more than that game. We had cooked ... sambar with drum-sticks and either of us was enjoying that with fried chicken. (now how does that combination go about ...eww/or Bhari, we were not in condition to make any discussion out of that). I was hooked to song “Iktara” from Wake Up Sid, a movie which fears fully resembles my past, irresponsible behavior. And m sure that those who had know me in past would node with a affirmative yes.

(you got ask this to : Tejas or Smruti or Swati or Pady or any friend from Apte college like or Asmya or Bhagya .. I subconciously still feel that chaayala, thoda abhyas kela asta tar ... hehehe pan .. ASO )

“ …. Ooh shit (“Shy” from: shy guy and “H--it” from : 10dulkar hit that ball for four ..type… Shiva would pronounce it like.. shY-iT ), dude its your birthday today I completely forgot you told me its in October… oh M so sorry man.”

“Its okay dude, its not the biggest day of the world you can miss that, devils are born with uncertain time and date.” I laughed.

“Paata nahi ..... kya baake jaa raha hain, hamesha bakwaaj karta hain, (don’t know what stupid thing you are talking, you keep dwelling into you outrageous things)” Shiva bashed that one on me. “Aapne pass to candle bhi nahi hain, (we even dont have a candle), no probs I will manage”.

I had a half ear to that and way back to the music. I wanted to mix the rhythms from a song from a movie Music & Lyrics (Hollywood movie) with a song call Maula mera Maula from a movie Anwar (Bollywood movie). So I just heard his words and left him to make some action on it. In next few minutes he was on his feet and showing his creativity. To be honest to myself it was brilliant on that line. He took a plate and placed some Sweet which he had brought recently from homeland and pierced it with Marlboro cigarette downside up. The packet was lying around for long untouched and unattended. It also needs a company to be inhaled and the climate or ambiance, which had to happen since I shifted to Shiva´s place.

He quickly got on his feet, went the door across, leaving two to three apartments, knocked Avinash Jr´s door. IN a while they both poured back into the room, were in audible range… and I could hear Avinash " … toh fir kya?", (so now ? I assume they were figuring out how to celebrate without a candle or maybe something else).

Avi (maharashtrians have this famous art of cutting the word in between, without making sure whether the person would like it or not, so it was Avi for Avinash. As it happens for me, "Parya" and nonetheless ... the name is always taken with utmost trust starting the sentence as "Aay Parya ...kay karto be" ), appeared in front, I removed my headset and then he gave me friendly hug and a firm shake hand of a cricketer. “Happy Birthday Mitra”(dost , friend), we wrapped our Marathi conversation in short and then we lighten the Shiva created “cigarette-sweet” and they sang hoarsely. We followed the usual ritual of blowing that and played some video on Youtube !! .....

- Parya

A View Point !

2009-10-10T09:15:00.005Z

Sometimes people gets sick with the 'everyday' people around them..but i believe they dont get tired and stop caring about them.

Its like when your in a relationship and you feel like its falling apart, you dont stop loving the person whose breaking your heart,the person you've been loving all your life just because of the stupid fights and arguements your having..

One has to step down coz it´ll never work if both of you are extremely mad with each other for a little stupid reason, or no reason at all..

We lose control sometimes of our feelings not knowing that someone is hurting,the fact that you, "not knowing" hurts even more.. When you really love someone that much, u´ll accept that person whoever,whatever she is.. and u have to accept the ones she loves as well..

Coz being a part of her life ,means taking a risks and sacrifices.. if u couldnt handle her fragile heart with care, then dont make it hard for her and just let her go as soon as possible

Coz u might not have an idea how much youll gonna hurt that person.. SPACE or time off.... not a good idea. It´ll just make things worst. When u ask for a space, it means your having a second thoughts or "it" doesnt feel like it was used to, and the spark wasnt there anymore..

For one thing i know for sure, sometimes, when the person ask for "it", one realizes that she really never meant it, and feels sorry about "them'.

The reply she'll get.."im sorry too but u were right. we needed space,for good"

OUCHHHH.....the point is, you should think first before you speak.. in other words, be careful what you wish for, coz u might just get "it" all.

If you dont want to experience heartaches and heartbreaks, you should learn to trust and respect,forgive and forget,and love while you can..

THATS ALL.

Chapter 5 : Flashback Reappearing !

2009-06-30T20:46:00.018Z

Chapter 5 : Flashback Reappearing !

“Screw you, man seriously … screw you!” Naga was impulsive making that obnoxious response on my behavior.

For the stake on my imagination, I was aware of his hating verbal. Not that I don’t find it offending but it crossed my mind that he was young and good at heart. I was wondering about the company he had invited as Kirti was not a girl to win on a beer. The other friend, Ranganath was a man of word, charming, mostly a chatter box with amazing sense of humor. The bell rang.

“Oh, I´ll get that door”, I said and moved away with my eyes making steady contact with his. I could sense he wished to have the company which left a minute ago for her room. “I will get this one, why don’t you get the other” I stared upwards and blinked at him, making a flagrant remark.

The door bell rang again, I hurried to the door and opened it softly.

“Hey buddy wass-up, long time no see” that was typical Ranganath, in short known as Ranga, the killer. The name had its own history but mostly due to his flamboyant nature and out fashioned dressing sense he was called killer, sarcastically. “You made it till here, now that’s acknowledgeable” I was little a confused as I couldn´t figure out, was it meant with sarcasm or was it genuine enough. As he stepped in, he was followed by a company along, a smart and elegant, talk for a girl to be.

I stepped back and promptly opened the door till its hinges and gave a small smile to her.

“Hello” that was not half bad as I thought of her being gal. She said that with no reluctance but I was not sure to reply first or admire her beauty in first place. On second hold I had just puffed my lungs with the so called intoxication, so yes I found it resisting enough to reply with smile. It clicked me, as if I had known this face before or crossed her path before.

“She is Jennifer, here for a semester. Finishing up with her Master´s in Landscape Design. She got an internship in Muncih. Thats great eh!” Ranga was happy to introduce her to all of the others.

She helped herself stepping out of the jacket. She had all the adages picture perfect, between the dress which she had put on was mesmerizing. A low neck shimmering mangenta red colour, sleeveless one piece; hanging high on thigh, with almost her entire back exposed, featuring her curvature of the spine running down till the region of heaven. A thin necklace going with small round ear rings. Her ballons were raised high, a clear indication of a person who is essentially particular about her figure. A clean shade of light blue colour twinkling eyes, a neatly done eye brows tappering towards the ear and a short but straight nose line with perfectly thick; sensual lips. She wore a complete thousand watt smile with those teeth pre caged with excellent alignment from one end to the other. A slight dark circle below her lower eyelid, almost hidden due to her black eyeliner, sure she must be a nocturnal creature. She must have been a good kisser and wild in bed while making love. Those legs stretched out, shaved and tender skin exposed. She was fair enough compared to most gathered there. Her collar bone was prominent and so was her sporty slick abdomen. She was a classic example of a high prize destination.

I have a theory for such person, who tend to desire more from their life and they believe; that – Change is Constant. As of me, if I had to define her beauty, then it would be the unblemished summation of the parts working together in such a way that nothing is needed to be added or taken away or altered ... Isn´t she was worth getting killed for.

Though her glamour was lovely, I sensed a negative vibe from it. Just an instinct kept me away from getting involved in conversation with her.

As the party got loud, I was getting high. I had a switch from beer to single malt, Glenfiddich. As I got more company to join me for bottoms up, glasses kept on getting emptied. Few of us got a hold of a corner and were enjoying the party from the farthest end.

“Hi, so you guys are straight upto whisky, that’s not half bad ....” a subtle voice from behind me joined the group

“Hmmm, saving the wine and beer for you” replied one of the member, smiling at her.

“How about you, you look totally wasted?” she directed that one towards me.

“Hunh, better than you I guess” I made it clean, and all laughed on that.

“Hi, Jennifer, Jenny ...” she re-introduced herself and left an ear for me to reciprocate. I didn’t, only a faint disinterested smile. She had a highly seductive accent and an erotic perfume; I could take that one home. Her glass was almost empty. “And you ...”

“Whatever you want me to be ....” and I was back to the glass.

“Hmm...Nice try” she whispered. She looked at my empty glass “You upto next ...?”

Being a gentleman, I took the lead “Need a refill on that?”

“You gonna gimme company for that?” again with a devilish smile.

A question answered in form of a question, I like that; being a smartass. I smiled and gave a thought to it. “Which one suits you?”

“M easy, though” she was reckless in answering “... but if you don’t intend to take me home then I would prefer Château de Villeneuve for me.” She had something going.

My eyes almost lit up “coming right up” I said and vanished in next.

I disappeared and made it back with double the speed. Somehow the comfort level was not upto the mark. She was sipping and making short conversation. My replies were rather short, abrupt and non-interesting. I pondered all over body taking X-rays from time to time.

“So you liked it?” she asked me genuinly. She saw me nodding affirmatively, I took that one for wine. “ ... I was asking you about the dress.”

I had no clue how to reply on that, so I gave a quick and hasty replied in low voice. I couldn’t process what I was saying neither did she.

“What´s that?” and she moved a step close to me so she could finally reach me in words.

“Nothing, I said you look ...... gorgeous.” This time she was so close to me that she could feel my eyes ravelling over her.

“I will take that as a compliment” she said. She had the look checking me inside out.

I raised my eyebrows and was sorting my thoughts in my mind. I was little confused as she entered the party with Ranga. So, was she an escort or just a friend! or maybe a friend with benefits? I could deal with the benefits but definitely not a friend. She was an ice cube too hot to handle. She was like a winner’s trophy to be taken home at the end of the day and left to be admired for the rest of the time.

“So what brings you here?”

I was hesitant to answer that initially but long thought, we would never cross the path so a little lie won’t hurt either of us. “Just roaming around in Europe, indulging my passion about photography, staying and travelling places” and I was slipping back to myself.

“Wow so less stress more party is it? All party and no work turns jack into jackass!” she ripped it off as she took the hold of the conversation.

That was hard enough to take, so I set it free. As I had nothing to hide that she would know of or vouch for. “Basically studies, which I don’t like to do, but yeah got to do ... no escape you see” I made it sound like I wasn’t passionate enough.

She was closing on me, “ ... N what would be that in your case.” She gave a look from top to bottom as if one is not even worth of a salesman. Her dialect seems to be playfully harsh. “Those broad shoulders, dark brown eyes and reckless look of your´s and with ladies professors around, you would fetch more than what you actually yearning.” That was mischievous and little notorious.

I was taken aback but she was smiling and enjoying every piece of her dialogue. I was unable talk her through. She was making me look foolish, so I thought to enlighten her, more about what would be esoteric for her. “Actually, m in a graduation programme.”

“Which one ! Hmm ... they gotta have some standard..... or”

I felt a relief as she started the sentence and then she came up strongly again...

“ .. do they have one? Anyways, good for you, different country and as you revealed you are not much for studies, so one more wallpaper to hang” She referred a degree as a wall painting. As if one could hide the cracks of wall behind those frame.
She was now turning me into complete idiot.

I was resenting my little lie at the start.

“Biology, something which is intricately complex to handle for someone with a beauty alone!” I had to give her that. She was smart but not much when it comes to play on the ground of brains. I wish I could have been correct here. Not only she left me dazzled but caught me on a blind spot when she had hit in reality in next conversation.

“Ouch, did I step on wrong foot”she clicked her eye and made it sound as easy as she could.

“Anyways, so how’s Deutschland treating you, made any friends around?”

“Aah, mostly either all I find are geek or too low to get along with, but you aren’t that bad. You wana get out here right now”

“Hmm, no I will pass.” I peered it away.

“How about you get me out of here and I let you take advantage of me, got a decent apartment, a short drive of twenty min from here”

“Lemme think , hmmm, how does NO sound” I had a mix feeling that she belonged to Ranga or maybe not ... I sensed something was coming up, something more to add.I was was much repelled to the talks.

She glanced around; she was making some eye contact. Introspection of some congenial material. “It’s pretty late, I think I will get going, wana help me get a cab. My cell is long hard to find, can I use yours” she wasn’t even asking, she was demanding instead.

“Sure, here it is ...”

She dialled a number, made it look that the other party on phone was not getting her due to loud noise. She moved away and returned late. She returned my cell“ yap, lucky me got hold ofone. So, biology, tell me is that only fascinating factor or you more into physiology of X” She switched the topic like her tongue twister.

I was surprised to see her profound knowledge and those remarks placed in those words. “I wish I could say something help you understand, but you had wine enough to get you a sleep”

“Yes, but another won’t do any more harm, may I ... ” She took my glass as well and returned with red wine. While returning the glass she made a observation of my fingers. She loosened the glass before I almost reached for. She left it purposefully, though I grab it before it could paint the carpet below. I had to bend down to make that happen. While straightening up I felt a hand near my neck and a small prick. Either the alcohol was ticking in or something else happened of which I was not aware of.

“Easy dear, you sure you wana drink that. A glass of wine left on its own certainly won’t harm either.”

“Thankx for your concern, I can take it from here. Gee, You seem to be acting like a soldier.” Her eyes were jovial, for what she was about to say.

“Ooh yea, good to know that, don’t give me that look m still not going with you. You can play good cop – bad cop stuff with somewhere else.”

“Soldiers are in army my dear. And besides army people are. They look masculine, with their weapons. They look more sexy. Much loyal to their company. Mostly, man of their word. M sure you got the correct weapon below that I can play with for a while. No I mean really, did you ever handle a weapon, apart from laying those hands on small hand-grenades.” She tripped a small glance at her boobs. She squeezed them a little between her shoulders, elevating the upper cleavage line sliding below her neck. That was highly gross even for a class of a hooker.

In a way she was hinting me and carry me towards a misleading road. And she was touching those topics where girls tend to shed away. Definitely something was wrong.

“What do you say, patching off or sliding separate ways?”

“Move on dear.” I tossed her for final word.

I had slight tingling sensation running through fingers, I clinched my fist and opened. My eyes blurred for a sec, the vision before me stretched out but reappeared. I gave a thought on giving up a break on the liquor. Suddenly I was fainting, trembling, feeling a pain in the muscle. Something like a drug has been administered. I was almost collapsing.

“Need a lill help soldier ... m totally available.”

“Sure thing, can you disappear on your own? I will feel much better.” My muscles were tearing apart.

“Don´t stress yourself, m here for your help, soldier.” She was moving closer

“You keep saying that word as if it means something to me!”

“Does it?”her tone was changed perfectly to an instructor.

“You tell me.”

“You sure about that, Captain Siddharth.”

“What?”

“Does it sound familiar. Same person with two different names, with two different identities. Except one is eradicate and one has been recently born.”

“Get away from me.”

“Keep absconding yourself from your previous part of life and it keeps getting worse. So many questions but no answer, right? Siddharth, don’t relinquish now, you aren’t getting any far. Sorry to say that.”

“What are you talking about?”

“We can do this here or we can take this outside, you need to take a walk with me right now.”

“Pretty lady, you are a guest here, behave like one. Remember you are here for a party, I belong to this place. I think you should take a walk.” A sudden headache and lethargic feeling came over me.

“You see I have administered a minor tranquilizer right through your cervical vertebra when you grabbed that falling glass of red wine, previously. Look around at ten o´clock and three o´clock behind me. You see those agents; I got a company for you. Besides you need an antidote for that tranquilizer in less than twenty minutes from now.” She looked into her watch. “So why don’t you roll along with me. We can take care of this outside. I don’t wana hurt anyone, certainly not you. I have been trying to trace you since last two years. For the record, I have something which belongs to you. Oh crap, I will cut the suspense, two missing fingers from you right leg. Forensic guys returned them to the CIA, but they were unable to find your body precisely. Poor thing. The thing is, you got the best surgery done to fix your face but m afraid those eyes don’t speak lie. You got me ... aahmmm.” She wasnt finished ripping me yet.

She had a retina scanner run through my eyes the moment she entered the party. The device was as small as it was fixed in the locket of her necklace. No one can replace eyes. She left with my cell and fixed a GPS tracking device in that. She went through the scanner before she made a move to join the our small group at the corner. A small chip was embbeded behind my neck. It´s thermal sensor was small enough to be placed on the tip of a finger. It is regulated by body temprature and gives a constant thermal signal which can be captured through satellite imaging.

I was already in deep shit.

Chapter 4 : Sports Bar.

2009-05-30T14:36:00.044Z

Chapter 4 : Sports Bar.

The street looked quiet, not a hiss of sound. All shops around the street were closed, filling more of the emptiness of the street. Though one thing went unnoticed, the “Sports Bar” was open, neon bulb glowing; deliberately coloring the wall above the hefty door. There was an ATM machine opposite to the door, up in front. It was strange to see a deafening silence on a Saturday evening nevertheless the arrangement of cars parked along the banks of street, on either side was much confined as ever.

A black BMW showed up 10 min past 20hrs and two formally dressed persons with office ties got out of the car which was left un-parked, as there wasn’t any place for. They were tall and broad, with black Ray-ban, suited there masculine face and more over went with their dress code. The angle of cheek bone was prominent, clean shave and an army cut. The coat was embarking the rich prodigy, both gave each other a look and headed straight to the bar. This was a perfect opportunity with no bouncers hanging around the door to step in with no permission required. They took their turns, a deep look at the corners of the empty road and then entered the bar. One of them took his glasses off and flipped out his identity card as he approached one of the bar tender behind the table. He kept the card low to his thigh, percautioulsy to be flipped out for use if required. Before he could start the conversation he was trying to go easy.

“Two beers, please” one of the person gave a low voice call with smile to the person behind the bar.

“You people don’t seem ´round the place, bar policy, no services without knowledge to restricted people” the person behind the table made a reply with a smile, as he was cleaning the glasses. Other way around supposedly making sure he gets an identity check.

“How about, I pay you double” the guy in suit was on it instantly.

“And why would you do that, just help me with your driver’s license and we are through.”

“Oh, is it! Then I forgot that one in my car, how about this, will this do” the other guy in the suit entered the conversation showing his batch and identity card.
The batch read, Agent Chondor, Secret Service, Aid to DEA chief to PODS. The guy was a secret agent, undercover directly in conjunction with The President of the Dept of Secret Service, Aid to The President of United States .

“... Pardon me sir, for any inconvenience if at all I have caused” the bar tender was gentle on the tone, “which one should I bring up?” as he looked up he somehow managed to press a buzzer under his table.

“Can I get a word with you, a quick one ! See what I can imagine here around is that the place is very small to accommodate the outside parked cars, so" Chonder took a pose, gave a mystique smile "that makes me think, where the hell are all the pals from those cars are screwing themselves ?”

“ Sir, I don’t know what you are referring to.”

The other agent stepped in, Agent Phenix and took the bar tender´s thumb his of right hand and pressed it hard backward.

“You tell me instantly, am accustomed to such practices, come out with the “stuff”, now. You see this gentleman isn’t gonna hold your finger, he will break it apart. And off the record, he is very impatient, so why don’t we keep this low, as I can see people around here.”

“Excuse me, can I help you gentlemen!" another person behind the table entered the chit chat. The startling look in his eyes was not pleasant, a gusty smile. He wished to give the two agents a shot in the balls.

“No I got it all from here, will make them home,” blurred the previous bar tender who was almost about to pee in his pants, as his thumb was almost folded back to touch his wrist.

“Thank you, that was a good move, you saved yourself a bone. You make a noise and I will break your hand. Come off the hook right now, see m gonna say it only one last time and as politely as I can, with all humanly possible dignity that universe has bestowed on mankind, don’t make me ask you it again, that will cost you !” Chondor was harsh this time, confirm with his words. Being on top of it as he shrinked his eyelids, looking straight into the bar tender´s eyes.

“Okhhy okhhy … take it easy folks" the bar tender moved ahead and lowered his voice "you see that, he over looked his shoulder to his left with his eyes drifting towards a chart "we have a back door leading downstairs, where the poker games are played. Of which the bet at one table starts from 10 grands, table called as - BLACK JACK . All "whales" around the town show up after past twelve, every Saturday night. New card for every game, every time the same person hits the table for the play, he is never changed. The chips are made in North Korea, shipped every Friday. The delivery is taken exactly at 11 hundred hours. They come in with tight security in civil dress; a team is allotted to count them as soon as they arrive. The container has 8 digit alpha-numerical code which is changed in every delivery and only the person in charge knows the code. A micro chip is embedded in forearm of the delievery guy, it runs on real time schematics. Magnetic chip is set in such a fashion that it calculates the altitude, pressure and thermal heat around and produces a 12 digit numerical code, that is the HOT KEY. This key is installed as Vault bar code, backwards." He took a small breathe and swallowed his epiglotis, cause what he was about to release now was classified information. He further lowered his decibels, there was strain in his voice this time. "It further requires palm and finger print validation from the head of the security. A facial recognition software is installed inside the Vault. That area is highly restricted and only accessible for Mr.McKoy, the owner of this organization which primarily permits the cash influx. So the gaming chips/coins are counted and made ready for game at night. The cash is always counted fortnight to check for any counter-fit currency. There is a Safe Vault, which holds almost 200 mils of current transaction in cash, mostly the black money is dumped and revenue is stagnated here.”

"Holly shit, they are running a F-u-c-k-i-n-g Casino down there," Chonder said it agitatedly keeping the voice to himself, covering him mouth with his fist after resting his left elbow on the table. Chondor girl existed from Nevada and he could envision the Las Vegas Casino instantaneously. He made a brisk remark to Phenix and shook his head non-assertively, "NOT GOOD".

Phenix took out a photo, with his eyes pondering around making sure no one is looking at them from his inner suit pocket and showed him. “Can you identify any of them, help me here else I will hold you to accessory for the crime.”

“Why are you people busting me? I have nothing to do with it. Swear please leave me out of this. I will feed you whatever I know to the best of my knowledge. Please either kill me or take me along when we are done. Two of them on the left are big shots in the Pharma industry, next to it is a person who owns a chain of restaurants on Linder Street, followed by Mr. Herman on the right, I have no idea about him, but he is shmuck and powerful, that makes him all the way more dangerous. An arrogant bastared who has some dot com business, according to the state department he is high on the black list but has many political connections. This is the reason he is never behind the bars. That´s about all I could say. And guess all the people sitting around here on this floor are for in the house security for all off these people …..”

“Hmmm, that speaks for itself, so the lousy entry on the door.” Conder said, while he was rubbing his forehead with left hand and signaled Phenix to let him go.

“Let’s get back and put the team on high alert, we need Herman and those pharma guys tonight, lets nip them in the bud before they can find an escape,” Condor lowered his voice and nodded his head to the bar tender to come out to the table. As Chondor turned around, three gigantic bouncer were already behind hi.

“Guests are not welcome” one bouncer said, he had his sleeves folded back and black polo neck shirt had a special mark on right hand side corner of the collar.

Phenix went casual on tone, “guys we thought we could grab a beer and play a girl around, Saturday nights get lonely. Thought will ride a horse and get laid back.”

“You should have noticed that girls aren´t found on front desk, and surprisingly to me; you people don’t seem to me as beer guys, why don’t you take your ass out before we crack your head open” other bouncer said "...and disappear like ...." there was a short pause, while the bouncer looked straight into Chonder´s eyes , leaned forward and said the word slowly as if splitting it .. "Forever.”

Chonder got down from his seat, loosened the tie and unbuttoned his suit. "Hmmm now you see that´s bit of a problem", his broad chest flexing back from relaxed posture through his lungs, attitude towards unborn bad people was aroused suddenly, “I was thinking of more on the line of single malt whisky, so boys can I buy you beer! We can get to know each other better with beer" Chonder cleared his throat, switched his tone to rock " and yes, of course if you are guys then lets get down to whisky on the rocks`, do man to man talk and get over with this ASAP." Phenix could see the whacky humour coming up next, "I wonder you folks must be under paid looking at your muscles, having loads of time on your hand to work on those and rest quarter of time, mostly wasted. So what do you say guys?” Chonder accomplished finally putting his hands on his waist.

Phenix immediately got down from the seat and mingled as he knew what Chonder was capable of, “guys guys … as we speak of we were about to make a move and get away” his voice showered to those entire bouncer and signaled Chonder not to enter a fight. Phenix shook the hand of a boncer with pleasure. He placed few bucks on the table and pushed Chonder out of the bar.

“Chonder, what were you upto? Are you nuts, you got cool down pal”

Chonder was an Ex Marine guy, tall, handsome, shade of blue eyes, fortunate enough to make any lady go weak in her knees. Unreasonably loyal to his Service, Military Core and Country with not a single scratch of doubtfulness ever. A decent head over shoulder; served his country and had restored for an inactive duty. His father was in army too. His inactive duty status was on the options, the country uses only in emergency. As a Captain in military he was involved in hostile recovery operation, but in Iraq it was different when he was recalled for immediate active service. He was back but never talked about it. He had been long dating a girl in Nevada before he was shipped to Iraq for the situation but then he never turned up to her after being back from duty since last 3 years. Since then he had problems with sleep , so never went home to his mother, infact his mother never knew he was back from mission. Was awarded twice, but never picked up any of them. He believed that those were steps to keep him away from his duty. He lost too many ranked officers and soldiers in his command, his memories were hardly any vague, which kept him awake in bed. His strategies in war were excellent as he once risked upon himself and his patrolling party. Brilliantly penetrated the plot and rescued the reminiscent soldiers and many Americans which were held hostage. Though at the end of war, the massacre was enormous; bodies of soldiers kept on coming in bags and flags mostly from his battalion. Practically, two lieutenants, one messenger guard and he himself were the only survivors of his batch employed on that mission. He had to call an air strike on the given co-ordinates as his options were exhausted. His fellows and two team officers were deployed and the curfew was still on, it was evident that it could have led to cease fire, consequence of which would have manifested to the end of the operation. As he had to called for an immediate air strike so he could at least got hold of few lifes but his major frustration was due to loss of his men. Saving life of politically powerful civilians made him hero but since coming day he couldn’t stand the mirror. His brain got screwed really bad, though his high rank military chiefs knew he did what was best in interest and they owed him big time. Inspite of all this, he resumed his inactive duty status back on the soil but he was shifted to DEA department as undercover agent. His file and core valid number was very confidential and classified. But he gave a damn to any of the state business.

“I don’t get you at times Phenix, it was such a great opportunity to massage my hands and get few swings for the shoulders, haven’t had this for a long time.” Chondor was passionate about fights as if he loved to beat the crap out of the bad guys.

“Get back to the car” Phenix knuckled him, as he saved the driver seat for him, revving the engine. The bouncer watched them fade away. “You know pal you need to get yourself some rest, this isn’t helping. You tend to forget that you are protector of law and not executioner. Get your affairs in line; you are not on military assignment right now, so don’t take matters in your hand.”

Chonder, looking straight ahead from the front glass of the car, smiled back to himself and pressing his lips as if the he had some thoughts coming up.

“Yes, how dumb of me, it must have fallen on deaf ear” and gentle tap of fingers with right hand on the car steering with left fist gripping hard, as he knew Chonder isn’t gona crack. Chonder was damaged to the edge, as according to Phenix a guy who denies sex is either lost his balls or insanely in love. Though only this theory goes for a toss when it comes to Chonder, because he was helluva commander of his mind.

Chonder, looked in the mirror with no fear what so every left inside him, he was about to say something, but then he kept those words to himself for now.

“As far I am concern, you were about to blow the cover, lets report pronto. There seems to be a larger movement. I have got eyes inside. Also took care of ears, planted with an IR device underneath the seat of steel, fixed stool.”

The eyes, the nano-chip surveillance camera was implanted below the table facing the door. Ears are similarly a small black magnetic chip which can catch sound from 120 meters.

“Good work, and yap thanx for the advice I will consider that, better luck next time ….. Roger come in” as Chonder gave a pat on Phenix´s shoulder and neglected all the talk advised to him. "Roger come in, Roger ... " Chonder picked up a black wireless handy, pressed the black button with his thumb and spoke into it.

"Hello, Cypher, Cypher leave your message here, Cypher ... We can hear you, please leave your message," a third party post replied back.

Chonder pressed and replied on the talky, "The Circus is about to begin, get the animals ready, we go IN, within next 30 mins, Over and out." The message was a signal to STF (Special Task Force) and SWAT (special weapons and tactics) team to get ready. The mission was about to begin, and SFT and SWAT teams are ruthless like animals in rescuing the given task.

Message was obviously cryptic, Chonder got rid of his glasses replaced his suit with bulletproof jacket, reloaded his riffle with a new magazine and checked his handgun for the bullets, `Let’s have fun boys `, and his eyes got sharper and overjoyed as this thought raced his mind.

Unlike anyone, Phenix had the slightest idea about Chonder because they were partners for last couple of years.

Chapter 3 : Getting wasted

2008-08-10T00:38:00.013Z

Had been worked up the whole day, now at end of the day sitting around the bench. I almost refused to answer myself on grounds of self-incrimination. None of the constructs was looking bad in sequences or other wise on the paper, yet there was no expression in vivo. It was getting hard to appreciate the work which was performed, did it made any sense. The semblance of work structure was profoundly embarking that hell lot of energy and dedication was put in last couple of weeks. It was getting bit annoying for myself with no conclusive results in hands. Knew that Reinhard was not gonna get wild at me but he was expecting more from me since last two months there has been exclusively no progress. All about I can think was to give myself a party to night, hang around with folks, get rid of work and free my already preoccupied mind. Oh no, on the second thought, stay back work on what is not analyzed yet, discuss new strategy with Kazu. In middle of all thoughts bouncing on top of each other, starring at laptop screen, trying read nothing and lost in myself.

“You aren’t coming, are you!” Few moments passed by and he was still standing around my table. “Hello is someone dead, deaf, mentally retarded, lost in no mans land, thinking of a girl on weekend! Hello you idiot get your ass out.” Still no reaction from my end. “Dude we also do science, so no need to pretend in front of me, try impressing people on your orkutlist.” Sagar was pretentious as usual.
I looked straight towards him from my sitting position with hasted deep breath inhaled.

“Aah that caught your attention, didn’t it” a muck smiled on his face.

“Yes, m coming with you, Sagar” I was plain and hard on his name, truly minding my own business.

“ You are not doing me a favor my dear and off the record you need to tell me whats going on at your end. Kuthlya pori cha laffda waagare nahi na !!! Nahi mhanje … bhabhi ko pranam bolna mera (did you land in getting caught is any girl stuff or did you made GF, if yes then give her my regards)” again with that mischievous smile .

“Let me get back to you in... hmmm…. say 15 mins ? got to go, see ya” I sprang up on my feet and in no time headed to Kazu´s cabin.

“But … ya … I mean, what m I suppose to do around here, hey listen … you get back here ASAP .. PARESH …” he was hovering and loud on particularly last word, but I disappeared in no time. “I can surf around you laptop, discover how cheesy you have been for last two weeks.” By that he meant he would go through my recently visited websites.

Sagar was new chap, famously known as Sagrya. Since last couple of months we had come close to know each other. As a matter of fact he landed in Munich and knew no more than one person which was apparently me. As he was reluctant in letting him know about himself, it didn’t bother much to me. Basically from Sweden, came over for his masters thesis an year ago in LMU. We often had visit to each others place for a drink or so. It was not until recent we happened to land in same institute, only the difference was he had completed his degree and was only looking for a position and me into my thesis. He knew how it sucks when results arnt coming. As matter of fact he never asked about work. We use to share simple talks while traveling, catching up with Indian politics, latest Bollywood movies, cricket and mostly of his friends back of Sweden. It was hard to predict him at times, extremely practical yet caring and astonishingly no strings of sentiments. Head strong, soft in speech, no ruggedness in behavior yet humors every time. He knew his boundaries all time, that what amazes me long time. Not a blurr on any subject, which was sensitive. He use to manage knowing things, how I still wonder, what more appreciable is his neutral attitude and low anger range. Low at its melting point but high at its boiling point.

“Kazu, is there any chance that we can replace the cassette of sequence which glows in red by the green one, something like GFP, which has range or 410 to 420 nm.”

“ And why would you do so, it seems that you are getting nervous over results, are you ? Since when did you started getting hassled.”

“Its just that m not so sure whether everything is in frame, cannot see fluorescence and therefore we can conclude no expression. Nevertheless the sequence looks good. Need to get through with this problem” it came with agitation.

“Oky, lets not jump on conclusion, lets go through it tomorrow afternoon and yes bring your protocol book, need to through you test digest for the constructs you injected. Foward me the MWG sequences of UncVenus gene. So we sit together around 14 hrs tomorrow ?” Kazu is always certain in his timings.

“Done then, lets finish off this, its hammering me inside. See you then” I proclaimed as if I had everything favoring me.

By time I returned to my place, Sagar was still fidgeting and had already screwed the Mac. He was trying different keys to open something, couldn’t find the button to close those application. The screen was messed up with almost every thing opened and he was lost in coming back to normal settings.

“Lets go, else we will miss the bus” I said to Sagar.

“ Wow Parya tuzha mac amazing ahe re (wow paresh your mac is amazing)” he was trying to cover up his ass of making mess on the mac.

Nikalas, next to my bench gave a satisfied smile hearing and picking two words of sentence, - amazing and - mac, “yap its truly fascinating.”

Arre, Y… Zzzd, tuzhya nanachi tang … koni kadhi mac getla ahe ka, ek pan “key” [keyboard keys] tyachi, dhad kalat nahi (you irrational-stupid fellow, who told you to get a mac laptop, none of the keyboard buttons are recognizable). Ugach shinnying maaraychi. Aapan kiti bhari dhakavacha (for no reason you want to act as smart ass and wana do show-off). He addressed me with his sarcasm. And on top he smiles at Nikilas as if admiring on his supporting statement. Left me confused and with no choice other than smiling at Nikalas and back at him, indicating Sagar for his pleasurable words.

“Can we move now or you still wana praise more, I can stand few more words” getting pissed at him.

“Oh, no its oky, I will spare them for next time, tell you seriously dude, my sentiments on this, `Good for nothing`” he was master in bashing anyone.

Switched off the mac, all application were of course forced quit without an option, no hesitation, it was all abruptly done. Leading the way out we moved towards the bus station. It was still warm outside at 7 in evening. We were back on our old topic, cricket, discussing stuffs as if we were the chairpersons with strong influential hold in Indian cricket team. In between my cell rang, I didn’t notice until Sagar told me. I looked the screen and smiled softly picking up the call.

“Dude you coming to cricket, its weekend and I know your must be working hardly,” typical of Naga, full of humor and enthusiasm.

“Actually, on my way home, need to cook and do some stuff” answered him back, “then need to wash my clothes and clean some stuff… and ….then you know …”

“Anything more is remaining to add in your perfectly , clumsy and useless life like .... you want to paint ! play your keyboard ! chat with girls ! call your parents …….” Naga hitting me, with his witty sarcasm.

“Oky, tell me what is the plan for evening” I did cut him short.

“Lets get drunk, watcha dirty movie, then cook chicken, which you can do for me alternatively, pub; where you pay for my drinks, what say you !” he was on it without a break, as if I was saying just that its his voice.

“ I will go with previous one without fail.”

“Yeah good boy, then you wont mind getting Hähnchen boneless from Aldi or Penny, which ever is near and convenient for you, would you!” the tone of his was so reckless making himself sure that I would do the task.

“Yes Prime Minister, with pleasure, anything else” smiling and enjoying our conversation.

“That’s all for today, will let you know if anything pops out, and ya ya listen listen … get Hells, eight bottles.” Naga likes to get spellbound after he is drunk.

“ Eiighhhhhtt …” I was stretching on the word deliberately.

“Ya, we got company …. my chief of staff and deputy director of communication dept … are on there way” Naga replied instantly.

“Was nice talking to Prime Minister, between who?” I had lost track whom he was referring.

“That would be Ranga and Kirti”

“Feel free to command more, it would be honor to serve you” I was making him more pissed at me by calling PM.

“You made your point validly, now get your ass here soon, we can waste better together, and for GOD sake stop this PM business” this time he was pissed on later part.

“See you then in an hour or so.” Putting my phone down.

When I was out on street with no place to get my head low during the new appointment of hostels, I needed a buddy to trust for dumping my "stuff" and guess what I got "Worth Of all". We started with urge of learning python a computer language. Mike is known as "NAGA: busy than prime minister of Munich. He calls himself "Sydney Brown" as he did his Masters from Queens College, Sydney University. Call him and he says he will get back to you and I swear he never does, I wonder though God stretches his day for 36hrs,could he manage it ! Though he is still juvenile and kid from heart. All time blasting humor, down-to-earth, gets along peers easily, bumps on making new friends, multitasking, manages to play cricket. Takes pride on his silly one-liners. Let goes indignation, lately stabilized, emerging to be sensible. At parties he is wacky and spellbound. He says "Sab Beer da Kassur hain" (its all because of beer). Becoming a workaholic, an adage is his new "Laptop". Classified signature, ever charming smile "bole to gOOd hain, ooya i mean to say gaane wala" (he is single and still straight orientation , sweet like molasses) .Loves one movie definitely Dil Chahta Hain (a bollywood movie blockbuster of 2001, out expressing friendship is divine) a lot. He is perusing his PhD from MPI biochemistry. In short he is called “Da.”

“Daru ka Parya, aaree leka tuzhya postmortem madhe –hardest part, liver asel, kadhnar ahes tu ticket over weekend drinking.”(drinks again. paresh, your postmortem report would identify the hardest part as liver, you are on verge to get a ticket to GOD due to your drinking habits of weekend).

We got onto bus and reached Am Hart. There we changed to U2 at U-Bahn for Sendlingertor. Further taking U6, last change over to our respective home, Harras and Hadernstern. Sagar got down at Harras, requesting me to keep the weekend alcohol low. I continued in same Bahn, within next ten, reached my destination. This time choose TENGELMANN, one of the most recognized foody stores over here. Its expensive a bit, but you get quality stuff.

Reached home in next 5, dropping my bag got, switched on the laptop. The OS opened but couldn’t log in. Aah shouted my head “SAGRYA”. He was a windows OS lover guy and mostly hated other OS for being non-user friendly. This time he had messed in USER terminal command line, so my log in was not recognized. I had to decode the error. It took almost half an hour, restarting and rebooting with recovery dvd, giving original license key. By the time it was almost 20.30, Naga stays five minutes by bike distance from my apartments. I went over the reading, putting the laptop on simulation, took out the plates from my bag and stored in the fridge. Telling myself, you got to run the PCR(polymerase chain reaction) and column purify (purification technique) for the plasmid tomorrow, no one is gonna be in the lab and that’s best time.

Intimately grabbed the keys, tumbled for a quick shower, had change and moved out with food stuff in bag-pack. Picked up race bike through low gear, bending over the handle, enjoying ride and iPod plugged into my ears screaming “Mayya Mayya” (item song, upbeat, from movie Guru). Sky was shaded crimson red with half brush painting white clouds, gentle breeze of late evening and girls strolling around the hostel, it wasn’t disturbing as it was still bothering what could have been wrong. By the time reached Naga´s place iPod was playing “Mungala Mungala” (very old song , extremely famous for drinkers ). It was just getting begun. He was living with other Indians in the apartment, all different rooms, sharing common kitchen and hall. I left bike unlocked, went upstairs, lower apartments that big house was fro his landlady. Rang the bell.

The door opened and there was “Hi” , was sweet, jolly voice and definitely an interesting person to know for a noble cause. I haven’t come across this fair girl or lady; I was not so sure before I could make any mark.

“Hey you ! didn’t they teach anyone in school to call up or ring the bell before they enter other privates property” Naga throwing at me.

“Firstly, m invited by PM, next I rang the door bell, I m sure you must have failed to notice that with such a company around (look at the lovely girl) and lastly they don’t teach how to call or receive calls in any school that are known to me.”

“You are wrong, except that you are totally correct, but you see, I might also be engaged in some private meeting in near future so what I was saying you must learn new ethics and act more civilized, also you …..” He paused and look at me, he could notice that he was only one standing and facing the door, his conversation was left unheard making him realize that he was doing monologue and I had already entered his house. He turned towards me “Okay, did you get those beers and you gonna cook for me right ?”

“Its in the bag, do you mind putting them in fridge before we gallop them.”

“Okay, I will do that, between she is Nivedita, my new housemate, new PhD” Naga said while making a move towards picking the beer.

I faintly smiled her back and got cigarette pack out from my pocket. Looking that she made no point of showing any further interest of her introduction to me.

“You guys enjoy, I got to catch up with some work” and she departed for her room, walking on upstairs.

“Man you need to speak, be more approachable, and cigarette in front of her, so gentleman of you na. Did anyone tell you that you are the craziest fellow I have come across” Naga unleashed his temper in low voice.

“What! she is like …. what your girlfriend or what ! No na, then why the hell do I care. I had been calling you whole last week, no return calls” I fasten on that.

“You are my girlfriend or what that I should call you back before the day is over, or leave you message in you mail box. Buddy there is rules you observe in front of housemates, you would care if you had one” he looked at me. “Presumptuous at core, that’s what you are. And you better don’t care for my girlfriend, save that part for me”

“So Drinking isn’t counted in the rules ? And for me not to care or otherwise you need to make one” continue the sentence while lighting up the cigarette.

“Hey have it ever occurred to you that you are never admired by anyone except I fail to observe that protocol earnestly (he smiled) and you ever open your cheap mouth its absolutely crap you speak, except your point are always significant. What the hell, who cares where we gona land up. So just hit it !” he wasn’t finished yet. Look straight into my eyes “by the way keep your dirt-looks.com and porn-thoughts.de, away from her” he pointed with his eyes upstairs at her room.

“Ooh ya, how m I not surprised about my unanswered calls now” I said sarcastically.

“You need to improve on that for bossing me next time,” this time I laughed at him.

“So what are you upto these days, leave that part tell me how is work coming along, oky chuck that we need to shop for today, remind me of that between did you bring enough chicken, we need do shopping pronto” he was smiling graciously “ oky I will let you think on all those, and put them to chill.” He got up and headed towards the fridge.

“I m fine dear, will update you shortly” I said buoyantly.

“That’s all I gonna get, man you need to tell me more, how is Nina doing? coffee? Call? Date?” with a notorious smile.

“Got more things to do, I m hooked up in lab for now, that’s more capitalizing for the moment, it’s the deal of the edge. To ensue of what is coming out is rather important than dating to 30-26-32. For your knowledge I have control over my jumping hormones”

“Screw you”

Chapter 1 : Darkness making the illusion of light !

2008-07-08T08:29:00.013Z

It was pitch dark with exceptionally no sound to disturb the concentration in that small room. Only the lens of Leccia, a 45K euro microscope with multiple computer operated functions, filtering out a strong narrow focus beam of medial light blue color of UV light. My eyes all set on the eyepieces. All happening in the moment, my guts didn’t want to believe it. After a long failure it was a sign of relief. In next second I pulled my thoughts back and I wanted my instinct not to believe the result of those injections. It was a bit of mix feeling, with subconscious mind being on cloud number 9 but with those socket of skull, eyes, bulging out ... making those conscious impose much brighter than light, awakening me.

Moving upright from the bend over aching, "Me" telling the "I " inside, “that’s disaster you idiot and you labeling the worst as your recreation as meaningful deed, but guess I should take some credit, why not!”

I was baffling inside, hmmm , so have I heard that good comes from bad, but whom I to decide on which end I m, all smiles across in mind. It chilled me through the spine as I was analyzing, all questions firing before one could jolt down the answer of previous one.

It all started few months back.

" So you interested in higher eukaryotes, I see" Reinhard was spontaneous "specifically in neurobiology ….. hmmmm! to tell you Paresh biochemistry is not what we do over here and you seem to be all hinged with those advance techniques"

Reinhard was on to that immediately with cups of coffee on either side of our left hands respectively. The only space between us, of the table was covered with few sheets of research paper.

He sipped his cup with 3 paged print out held in his other hand " Hmmmm ... quite impressive I must say" there was a short pause after that, he kept my CV back on the table. "So tell me again what brings you to my lab?” and he sipped again, waiting for my answer “work or just the desire to try new things" definitely he was in mood of an interview, so was I prepared for that.

" Well now that you have asked, let me be honest then, bit of both, include the factor which you left out to mention and that would be funding" I replied

" You worked at MPI Biochemistry with experienced and two leading groups. An independent work furnished with good output. You could have continued there as well, then why my group?" his eyebrows making all the talk this time.

" Zebrafish is excellent model for neurobiology, transparent as glass; a good see through body, easy to breed, cultivate and handle in lab, recently entire sequence has be put up on net, amenable to large scale screening , elucidating tissue specific gene expression pattern of medically relevant processes in humans, such as inflammation, angiogenesis, neuronal growth, bone and muscle formation and insulin regulation, most exciting is to have a working live model an in vivo looking at developing in front of your eyes" I spurted out as if I knew he was gona ask me.

With a flabbergasted look into his eyes "did u breathe in between!” an quenching smile “I m kidding. And you seem to taken extra credits as well for neurobio, n what was that for? Ah, Cellular neurobio, indeed" he continued "Oky let me quickly tell you what my lab is interested in."

Before he could almost end the sentence " Dynamic neuronal migration in cerebellum region, you have a working hypothesis for few neuronal disease including Alzheimer and Spinocerebellar ataxia, wana look into tumor formation, migratory pathway for developmental pathways, proliferation and metastasis." I exhaled out totally and took a slow deep breath.

" Which precisely tells me that you have studied my lab page pretty seriously and you have an internet access as well " he smiled at me " did u happen to interact with anyone of my student?"

" Nope, but I was smart enough to not miss your conference talk at Unvi on last Monday. "

And now we both laughed. It was good start with boss not loading you much of info on first meet but I was about to prove myself wrong on this.

" Lets get to the topic of interest then, ready! " He looked into my eyes with clear thought and nodded with affirmation." I m interested in a developing disease model in zebrafish on lines of the developmental pathway called Shh-Patch, called Medulloblastoma, most common malignant brain tumor, originally considered to be a glioma, it is now known to be of the family of cranial primitive neuroectodermal tumors (PNET), affecting children of age 4 with, 6-10% of all childhood cancers, mortality rate upto 100% with no medicinal cure as of now. What we know from previous articles is, site of origin is cerebellum, a region of the brain that plays an important role in the integration of sensory perception, coordination and motor control” he was about to pour more on it between he saw my face going blank. “ was it too much for the day to begin with ?”

Conversation went on for a while. He made sure that I understand things, so he had slowed down in between showing slides on his laptop of cell migratory pathway, fluorescence of cerebellum region. It was still alien for me as in I had studied in my class but not to this depth. Was slow percolation as he carefully narrated me through the entire work plan. I had hardly missed anything cause this time my all neurons were fired and elicited.

He showed people around the lab. An opportunity to talked with two postdocs Jen, a smart & slim and yet down to earth Canadian lady, Kazu a, Japanese huge hulk on his bench, a gentle and sincere smile at me. There were others around. Now the PhD´s; Katherin, German blond wearing a sweet smile, she was on her laptop, Nikalas, weirdo hair and perfect athlete stature the only guy who exchanged a shake hand. On the microscope playing was Matrin, almost 6.2* tall, handsome anyone could notice those blue eyes, was busy so jus a quick “Hallo” all mystified.

“He is Enrico, you may be around with him for a while he will tell you how things work around, color code, what is where and other stuff.” Reinhard turned and “ and she is Petra who looks into to administration part, you will need her for regulation and key part.” Both them with German accent making me comfortable saying “willkomen.”

This told me that I could be in a position to be selected, cause he was expressing his concern “would be likely, could be correctly, may be definitely in position to elect me in his lab” with an internal drift of feelings. We were back to his office, back in same position & place seated. “So would you like to join for this project, let me know, ya and how about your course work, when would your likely date of join if you make up your mind?” sounding boss this time.

“ I m yet to get through my course work, few to finish but that wont be matter I guess, I have few by mid of feb and I m through atleast for this seme. Would like to join may be jus after that, have no plans to flyback home” I was crystal clear about taking this opportunity as I knew this would be beneficial and freedom of work with postdocs helping me out in scientific discussion. Else was matter of money which I was desperate, I had to support my increased fee and stay over here. Without missing on this “would I be supported for this whole 9 month of duration.”

“ We do not pay trainees here, its against the institutional law” he looked at me straight and could make out an uneasiness crawling through. “Nevertheless, I will let you know if I could make some arrangements for you. I understand its tuff without getting paid, but lets see how could we fit this one. Oky then, hope, I can do something for you, will getting in touch with through mail. All the best for your exams.” He got up and made a firm shake hand.

I was delighted, making no mistake, I knew I had made no impression on him, but wasn’t dump enough though. In follow days he mailed me that he could take me as ´HiWi´ a workstudent as they say over here, in short `helpers job´ with payment decent enough for my sustainability. My seme got over and follow next week I joined the lab for thesis, it was feb.

I called up home to let them know this good new. What was I think with being so clueless and I heard voice back “ so you coming back after two years then” who could it be other than my happy darling mom, with plans in her mind all set before I intend to come to end.

“ Ya , ya … ya , ya mom, take it easy I m yet to join the lab and have to work sincerely and full enthusiasm. You making me nervous by raising your hopes to see me so soon.” Ahh I said, fool to myself, now closing my eyes “ M sorry dear if I hurted you, but I m here for education. I cannot be back before I finish.” No response from other end, she was sobbing. “ Oky, you know I don’t make promises which I cannot keep, I will try my best to be back soon, oky dear !”

“I am tired of your such unworthy understatements, you are acting all grown up. Go ahead make yourself successful and you don’t dare to fail in making me proud and don’t forget to take care of yourself” that was typical of her. “Your bro n sister in law is coming this December. Australia seems to have suited them. And you nuts make a point to come down during that period of time. Tuzha research kahi marnar nahi, nahi tari kay diwe laavto tikde kon jaane (your research wont stop or die if you don’t work for a while) ” mom with her ever jolly tone back into mood. “ Tell me are you comforting any gal or you still haven’t made any move, I bet my kiddo has been spellbound”

“You know darling, you love me so much that I am afraid, how will you do if I m shared ” I giggled. It was my duty after all to bring those checks back, nevertheless I knew she was smiling plastic this time. An instinct told me, she is expecting this baby of her´s this time. But I was helpless

The OC !

2008-04-08T21:30:00.002Z

'The O.C.' opens with bad-boy Ryan Atwood, finding himself in trouble- in trouble with the law, and in trouble at home. Against all odds, pro-bono public defender, Sandy Cohen, takes Ryan under his wing, and back to his home in Newport Beach, Orange County, California. Ryan finds himself immersed in this high-class society but soon realizes nothing is as it seems.

--------------------------

Ryan is in trouble after a police chase and his brother Trey goes to jail for 3 to 5 years.

Sandy is assigned Ryan attorney and gets his charges reduced. When Ryan returns home his mother in a drunken state kicks him out on the streets.

Sandy's business card in his hand and in one phone call Ryan ends up in one of the most exclusive coastal communities in Californina. Pool house digs and a whole nother life awaits him if he can keep it together.

Kristen who appears cold and less than warm about the idea of Ryan being in her home with expensive items at hand for him to steal agrees to a trial stay.

His son Seth being a only child will have to adjust and make a friend or go on living in the house a secret life his parents know nothing about.

Ryan learns to play the game and in the meantime Seth and Ryan may have just found a friend for life. Until the neighbor girl Marrisa complicates things left and right.

Summer who is Marrisa rich and spoiled friend is also a detour Ryan doesnt want to take but once Summer wants something all the money in the world has always solved her problems and I dont believe she has ever heard the word NO.

Luke is the all american guy everyone wants to be his friend and no one ever wants to be his enemy. He has the good looks and a attitude for trouble. Ryan and Luke will never be friends.

Throw in a Fashion Show with thin rich girls , Druken kids looking for a fight it seems for any reason , a few girls throwing themselves at boys and you got yourself a hot new show on FOX. But that is just the kids...the parents are a whole nother story you gotta see.

Greys Anatomy Season 4 E 11 (BROKEN - by Lifehouse)

2008-02-04T09:52:00.003Z

The Broken clock is a comfort
It helps me sleep tonight
Maybe it can stop tomorrow
From stealing all my time
And I am here still waiting
Though I still have my doubts
I am damaged at best
Like you've already figured out

I'm falling apart
I'm barely breathing
With a broken heart
That's still beating
In the pain
There is healing
In your name
I find meaning
So I'm holding on (I'm holdin on)(I'm holdin on)
I'm barely holding on to you

The broken locks were a warning
You got inside my head
I tried my best to be guarded
I'm an open book instead
And I still see your reflection
Inside of my eyes
That are looking for purpose
They're still looking for life

I'm falling apart
I'm barely breathing

With a broken heart
That's still beating
In the pain (In the pain)
Is there healing
In your name
I find meaning
So I'm holding on (I'm still holdin on)(I'm holdin on)
(I'm still holdin on) (I'm holdin on)
I'm barely holding on to you

I'm hanging on another day
Just to see what, you will throw my way
And I'm hanging on, to the words you say
You said that I will, will be okay
The broken light on the freeway
Left me here alone
I may have lost my way now
But I haven't forgotten my way home

I'm falling apart
I'm barely breathing
With a broken heart
That's still beating
In the pain (In the pain)
There is healing
In your name (In your name)
I find meaning
So I'm holding on (I'm still holdin')(I'm holdin' on)(I'm still holdin') (I'm holdin' on) (I'm still holdin')
Barely holding on to you (I'm still holdin on)
Barely holdin on to you

.. i agree !!!

2008-01-31T17:20:00.000Z

... i agreed to it do u ?

"The longer I live, the more I realize the impact of attitude on life. Attitude, to me, is more important than facts. It is more important than the past, the education, the money, than circumstances, than failure, than successes, than what other people think or say or do. It is more important than appearance, giftedness or skill. It will make or break a company... a church... a home. The remarkable thing is we have a choice everyday regarding the attitude we will embrace for that day. We cannot change our past... we cannot change the fact that people will act in a certain way. We cannot change the inevitable. The only thing we can do is play on the one string we have, and that is our attitude. I am convinced that life is 10% what happens to me and 90% of how I react to it. And so it is with you... we are in charge of our Attitudes."

So wht is it like ... !!!

2008-01-24T19:12:00.002Z

So wht is it like ... !!!

Some times u really feel like been known as a particular person and the next sec u wana be wht u R ..... Time flies by and u realize, tht u keep on appending every moment of ur life , jayala appan " SHAAN " asa pan mhanto, taking turns in wrong or correct directions .... there is change of place n ppl , which gets u upto a particular mark ..... Looking back , shows wht u were , looking in the mirror tends u to guess wht u r and finally in dreams u render to be " THE ONE " .... hmmmm ...

Simplest complication is most difficult puzzle , answer to which is rather very easy, jus being over thoutful and criticized by u own . ..... Few say time is best medicine , few say wht ever happens, happens for the best ... but hardly anyone realizes tht we R intimidate as driven by someone ... and i get comments back ..... CANNOT AGREE WITH U ON THIS PARESH !!!! ..... yes may be .... experience makes u believe in these conditions ...

Almost everyone , has gone thru this " we get up and some thing is similar around , u walk out of house and it seems tht u have gone thru this scene " havent u ? ..... U mesmerize and then jus let it go saying tht either u r stressed or recently u have been to busy . ... some name it as illusion ... but is it really so , is tht u could hope to see in dreams and then it jus happens to u the next day ..... MIND GAMES ! or CAPACITY TO have FORESIGHT ...

Think on this ...

Tare Zameen Par

2007-12-31T08:51:00.003Z

Kholo Kholo Darwaze
Parde Karo Kinare
Khuntey Se Bandhi Hai Hawa
Mil Ke Chhudao Saare

Aajao Patang Leke
Apney Hi Rang Leke
Aasmaan Ka Shamiyana
Aaj Hamein Hai Sajana

Kyun Is Kadar Hairaan Tu
Mausam Ka Hai Mehmaan Tu
O, Duniya Sajee Tere Liye
Khud Ko Zara Pehchaan Tu

Tu Dhoop Hain Jham Se Bikhar
Tu Hai Nadee O Bekhabar
Beh Chal Kahin Ud Chal Kahin
Dil Khush Jahan Teri Toh Manzil Hai Wahin

O, Kyun Is Kadar Hairaan Tu
Mausam Ka Hai Mehmaan Tu

Baasi Zindagi Udaasi
Taazi Hasney Ko Raazi
Garma Garma Saari
Abhi Abhi Hai Utaari

O, Zindagi To Hain Batasha
Meethi Meethi Si Hai Aasha
Chakh Le Rakh Le
Hatheli Se Dhak Le Ise

Tujh Mein Agar Pyaas Hai
Baarish Ka Ghar Bhi Pass Hai
O, Roke Tujhe Koi Kyon Bhala
Sang Sang Tere Aakash Hai

Tu Dhoop Hain Jham Se Bikhar
Tu Hai Nadee O Bekhabar
Beh Chal Kahin Ud Chal Kahin
Dil Khush Jahan Teri Toh Manzil Hai Wahin

Khul Gaya Aasmaan Ka Rasta Dekho Khul Gaya
Mil Gaya Kho Gaya Tha Jo Sitara Mil Gaya, Mil Gaya

O, Roshan Hui Saari Zameen
Jagmag Hua Saara Jahaan
Udne Ko Tu Azad Hai
Bandhan Koi Abh Hai Kahan

Tu Dhoop Hain Jham Se Bikhar
Tu Hai Nadee O Bekhabar
Beh Chal Kahin Ud Chal Kahin
Dil Khush Jahan Teri Toh Manzil Hai Wahin

O, Kyun Is Kadar Hairaan Tu
Mausam Ka Hai Mehmaan Tu

Tum Se Hi Tum Se Hi

2007-12-13T07:55:00.002Z

Na hai yeh pana
Na Khona hi hai

Tera Na hona jane
Kyun hona hi hai

Tum se hi din hota hai
Surmaiye shaam aati
Tumse hi tumse hi

Har ghadi saans aati hai
Zindagi kehlati hai
Tumse hi tumse hi

Na hai yeh pana
Na Khona hi hai
Tera Na hona jane
Kyun hona hi hai

Aankhon mein ankhne teri
Bahoon mein baahein teri
Mera na mujhe mein kuch raha hua kya
Baaton mein baatein teri
Raatein saugatein teri
Kyun tera sab yeh ho gaya
hua kya
Mein kahin bhi jata hoon
Tumse hi mil jata hoon
Tumse hi tumse hi

Shoor mein khamoshi hai
Thodi se Behoshi hai
Tum se hi tum se hi

Aadha sa wada kabhi
Aadhe se jayada kabhi
Jee chahe karlu is trah wafa ka
Chode na chote kabhi
Tode na tute kabhi
Jo dhaga tumse jud gaya wafa ka

Mein Tera sharmaya hoon
Jo mein ban paya hoon
Tumse hi tumse hi
Raste miljate hai
Manzile miljati hai
Tumse hi tumse hi

Na hai yeh pana
Na Khona hi hai
Tera Na hona jane
Kyun hona hi hai

Bas Ek Pal

2007-12-06T09:03:00.003Z

Bas ek pal mein- 3

Tu ek baar jo pyaar se mujhsko chuley tho har zakhm bhar jayegaa,
Zara ithzaa suun ke dewaane dilki mujhe aapne dilse lagaa...

Tere pyaar mein aise jiyain hum,
Jala hain yeh dil....yeh ankhen hue Nam - 2

Bas Ek Pal....... - 3

Hamare Khayalo mein Khwabaon mein yadhaon mein Bathaon mein rehthe ho tum,
Badaake mein yeh haath chuna jo chahuin tho pal bar mein hothe ho Gum.

Tere pyaar mein aise jiyain hum,
Jala hain yeh dil....yeh ankhen hue Nam - 2

Bas ek pal mein- 3

Sunaah hain Mohabbath ki Thakdeer mein, Likhein hain andherein Ghanee,
Thabi aaj Shayad Sitare Sabi zara saahi roshan hue...

Mere haath ki in Lakeeron mein likhe abhi aur kithne... sitam,
Khafaa Ho gayein hain kushi waqt sey ho rahein hain meherbaan Gam,

Tere pyaar mein aise jiyain hum,
Jala hain yeh dil....yeh ankhen hue Nam - 2

Bas Ek Pal....... - 6

Bas Ek Pal

Chak de India ..

2007-12-05T11:01:00.002Z

(kuchh kariye - 2) nas nas meri khole, haay kuchh kariye
(kuchh kariye - 2) bas bas bada bode, abb kuchh kariye
ho koi toh chal zidd phadiye, tu bidarayiye ya mariye - 2
chak de ho chak de india - 4
(now where to run now where to hide
lets take our time till we drop) - 2

poocho na galion mein, rashan ki phalion mein
bailon mein beejo mein, eedon mein dijon mein
reton ke daano mein, filmon ke gaano mein
sadko ke gaddon mein, baaton ke addo mein
humkara aaj bhar le, das baara baar kar le
rehna na yaar pichhe kitna bhi koi khinche
tas hai na mas hai ji, jid hai toh jid hai ji
pisna yuun hi pisna yuun hi paththar yeh
koi toh chal zidd phadiye, tu bedarayiye ya mariye - 2
chak de ho chak de india - 4
(now where to run now where to hide
lets take our time till we drop - 2
chak de.........

ladati patangon mein, bhidati umango mein
khelo ke melon mein, balkhaati relon mein
ganno ke mithe mein, khattar mein jhitein mein
dhundein toh mil jaave ? wo ? mein
dung hai ? nikharein aur khul ke aag bikharein
man jaaye aisi holi rug rug mein chal ke boli
tas hai na mas hai ji, jid hai toh jid hai ji
pisna yuun hi pisna yuun hi paththar yeh
koi toh chal zidd phadiye, tu bedarayiye ya mariye - 2
chak de ho chak de india - 4

ki aab kuchh kariye..........

... Keep Smiling !!

2007-12-05T07:34:00.002Z

Smile shows friendship
Smile makes new friends
Smile makes other people's day brighter
Smile improves your day
Smile looks better than a frown

Smile Puts others at ease
Smile always enjoyable to give and receive
Smile leaves favorable impressions
Smile makes you look happy, confident, and self-assured
Smile could be the start of a lifetime relationship!

So, my dear friend. Never leave the smile from your face!!

The Calling - Wherever You Will Go

2007-11-23T21:58:00.003Z

So lately, I've been wonderin
Who will be there to take my place
When I'm gone, you'll need love
To light the shadows on your face
If a great wave should fall
It would fall upon us all
And between the sand and stone
Could you make it on your own

If I could, then I would
I'll go wherever you will go
Way up high or down low
I'll go wherever you will go

And maybe, I'll find out
The way to make it back someday
To watch you, to guide you
Through the darkest of your days
If a great wave should fall
It would fall upon us all
Well I hope there's someone out there
Who can bring me back to you

Runaway with my heart
Runaway with my hope
Runaway with my love

I know now, just quite how
My life and love might still go on
In your heart and your mind
I'll stay with you for all of time

Reality

2007-09-21T00:37:00.003Z

Too often we don't realize
What we have until it is gone
Too often we wait too late to say
"I'm sorry - I was wrong."
Sometimes it seems we hurt the ones
We hold dearest to our hearts
And we allow foolish things
To tear our lives apart.
Far too many times we let
Unimportant things into our minds
And then it's usually too late
To see what made us blind.
So be sure that you let people know
How much they mean to you
Take that time to say the words
Before your time is through.
Be sure that you appreciate
Everything you've got
And be thankful for the little things
In life that mean a lot.

महि वे

2007-09-04T13:47:00.003Z

Jaan hey kya
dil cheez kya
mein tera sub kuch mera
suna suna mera jeevan
merey nazddek aa
mahi ve o teri yaad aati hey rey
o chandn see dhoop mein
o badli ki ot sey
mahi vey teri yaad aat hey rey
tu ik sundar pyar ka sapana lagey hey rey
mahi vey
mahi vey

hosh ho na khabar ho
ik aisa safar ho
aur hum saath hon
raat jugonno sitarey
rang khusboo nazarey
pyar key hon ishary
aur tum saath ho
suna suna mera jeevan
merey nazddek aa
mahi ve o teri yaad aati hey rey
tu ik sundar pyar ka sapana lagey hey rey
mahi vey
mahi vey

rooth jaaney mein kya hey
man jaaney mein kya hey
dil jalaney mein kya hey
ab tum jaan lo
mukhtasar hey fasana
mein hun tera deewana
dil na mera dukhana
ab tum jaan lo
suna suna mera jeevan
merey nazddek aa
mahi ve o teri yaad aati hey rey
tu ik sundar pyar ka sapana lagey hey rey
mahi vey
mahi vey

mahi ve o teri yaad aati hey rey
o chandn see dhoop mein
o badli ki ot sey
mahi vey teri yaad aat hey rey
tu ik sundar pyar ka sapana lagey hey rey
mahi vey
mahi vey

Flying Without Wings

2007-08-20T20:07:00.003Z

"Flying Without Wings"

[Shane:]
Everybody's looking for that something
One thing that makes it all complete
You'll find it in the strangest places
Places you never knew it could be

Some find it in the face of their children
Some find it in their lover's eyes
Who can deny the joy it brings
When you've found that special thing
You're flying without wings

[Mark:]
Some find it sharing every morning
Some in their solitary lives
You'll find it in the words of others
A simple line can make you laugh or cry

You'll find it in the deepest friendship
The kind you cherish all your life
And when you know how much that means
You've found that special thing
You're flying without wings

So, impossible as they may seem
You've got to fight for every dream
Cos who's to know which one you let go
Would have made you complete

[Shane:]
Well, for me it's waking up beside you
To watch the sunrise on your face
To know that I can say I love you
In any given time or place

[Mark:]
It's little things that only I know
Those are the things that make you mine
And it's like flying without wings
Cos you're my special thing
I'm flying without wings

[Shane:]
And you're the place my life begins
And you'll be where it ends

[Mark:]
I'm flying without wings
And that's the joy you bring
I'm flying without wings

Listen to your Heart

2007-08-13T08:50:00.003Z

I know theres something in the wake of your smile.
I get a notion from the look in your eyes, yeah.
Youve built a love but that love falls apart.
Your little piece of heaven turns too dark.

Listen to your heart,when hes calling for you.
Listen to your heart,theres nothing else you can do.
I dont know where youre going
And I dont know why,but listen to your heart
Before you tell him goodbye.

Sometimes you wonder if this fight is worthwhile.
The precious moments are all lost in the tide, yeah.
Theyre swept away and nothing is what is seems,
The feeling of belonging to your dreams.

And there are voices,that want to be heard.
So much to mention,but you cant find the words.
The scent of magic,the beauty thats been
When love was wilder than the wind.

Echos in Eternity ....

2007-07-08T20:27:00.001Z

TO SUM in Short : Carry away ....
Clean the wounds.
Stop giving explanations.
Make up Ur mind.
Decide and be determined.
Follow the path.
Dont look back, live the present.
Make friends for lifetime.
Lower the verbosity.
Open Ur heart B generous.
Help urself first.
Do no worng
Paint your picture.
B loud = C A U S E
" WHAT WE DO IN LIFE ECHOS IN ETERNITY "

PARESHVISHWASRAO

Into the fire ......

2007-07-04T21:45:00.002Z

Come on, come on
Put your hands into the fire
Explain, explain
As I turn and meet the power
This time, This time
Turning white and senses dire
Pull up, pull up
From one extreme to another

From the summer to the spring
From the mountain to the air
From Samaritan to sin
And it’s waiting on the end

and now I’m alone I’m looking out
I’m looking in, way down
The lights are dim
and now I’m alone I’m looking out
I’m looking in, way down
The lights are dim

13 senses : they rock !

PAATHSHALA-BE A REBEL

2007-06-29T21:18:00.002Z

Say hoi ho oh hoi ho (x2)
Loose Control
Be A Rebel

To the mahal of the Taj
To the Minar of Qutub
To the Kumari of Kanya (Say hoi oh hoi)
To the Panjim of Goa (Say hoi oh hoi)
To the Kaancheepuram
Zindabad Zindagood
All have something to say (Say hoi oh hoi)
They all have something to say
They all have something to say

Follow me Follow me Follow me now!
Apni tho paatshala, masti ki paatshala
Be A Rebel

Naa koi padne waala, Naa koi seekne wala
Naa koi padne waala, Naa koi seekne wala
Apni tho paatshala, masti ki paatshala
Apni tho paatshala, masti ki paatshala
Loose Control

India and Pakistan
Bangladesh all in the soul
Khajuraho to Coimbatore
Say hoi oh hoi
(Say hoi hoh hoi)
>From the Gateway of India
Upto the Himalayas
Across the Ganga Yamuna
Say hoi oh hoi
(Say hoi hoh hoi)
To Shirdi to the Red Fort
Rourkela Yeah u know
>From Ajmer to Orissa
Say hoi oh hoi
(Say hoi hoh hoi)

Loose Control
Be A Rebel

Amar Akbar Antony
Ram Rahim Abdullah
Searching for a bright star
Say hoi oh hoi
(Say hoi hoh hoi)
To the corners of the Earth
What is our (search??) worth?
What is our destiny?
Say hoi oh hoi
(Say hoi hoh hoi)
Imagine we go back in time
Free your soul, Free your mind
Take a look what do you find?
Say hoi oh hoi
(Say hoi hoh hoi)

Loose Control
One more time!!
Loose.. Loose..
Loose Control
Haha (x3)
Be A Rebel

Open it now .......

2007-06-26T09:34:00.001Z

Open your ears, make them hear
Listen 2 the breeze
Its making a symphony which is telling u something
Dont ignore it , cause it will come back to you.

Open your eyes, make them see
Scenes of past 2 unravel the cast
Dont waste time thinking
Pick up the needed and be ready to paint the next " thing "

Open your mouth, make it speak
2 communicate if necessary
Not to comment but 2 appreciate others
Dont blurr , be confident n low.

Open your mind, make it imagine
The future which you want 2 enter
Carve the path to walk on it , when u " rise "
Dont do wild guess cause it might land you in mess.

Open your heart, make it beat
4da B-loved one´s.
To stand for them at any instant
Be so strong that any strom should droop in front
Dont rush atal, it will fail if u R weak

Long is the journey , Stops are many
Some are immediate some are tiny.
U dont stop , cause u have to be " there n their " !
Drive , Swim , Run , Breathe but dont Hault .
Cos there is no End until u decide "

IsN´t that so true ?

-- Paresh Vishwasrao

My wish

2007-06-16T23:28:00.001Z

I hope the days come easy and the moments pass slow,
and each road leads you where you wanna go,
and if you're faced with a choice, and you have to choose,
I hope you choose the one that means the most to you.
And if one door opens to another door closed,
I hope you keep on walkin' till you find the window,
if it's cold outside, show the world the warmth of your smile.
But more than anything, more than anything...

My wish, for you, is that this life becomes all that you want it to,
your dreams stay big, your worries stay small,
You never need to carry more than you can hold,
and while you're out there getting where you're getting to,
I hope you know somebody loves you, and wants the same things too,
Yeah, this, is my wish.

I hope you never look back, but you never forget,
all the ones who love you, in the place you left,
I hope you always forgive, and you never regret,
and you help somebody every chance you get,
Oh, you find God's grace, in every mistake,
and always give more than you take.
But More than anything, yeah, more than anything...

LIFE !

2007-06-10T20:56:00.004Z

Hmm .....................
Its moulding the patterns.
Couldn´t be more close to know what,
I could have ever known.
To known what I ever thought of knowning ....

Tried to define the definition of
"DIFFERENTIALLY SIMILAR"
But ended upon knowning
" IDENTICALLY PERFECT "

Still the day is worth spending at work,
And get started with the new ray of hope ....
But, now I have done with the most which.
I could ever thought so......

Time is priceless.
So get up n get going.
Still new horizones , fresh air, new roads and melidious sounds
are to be explored.

PARESH VISHWASRAO

Words

2007-06-10T11:44:00.003Z

Expressin things in da form of words,
Some r soothin, some act like sword.
Juliet sayin - " I Love You " melted Romeo´s Heart.
Words of hatred n anger drive lovin-1´s apart.
Words of Consolation support the fallin-1.
Words of Appreciation make impossible things done.
Confession of `things` makes somebody´s life,
N words of ´hatred` hurt heart like knife.
Thus small word make difference big,
They can make u begger or make u king.
Will act of emotion be ever understood ?
Can unsaid words speak volume,
Much loud enough 2 b heard ?

Track the genetics : Drosophila melanogaster

2007-06-08T09:20:00.000Z

Drosophila melanogaster

Male Drosophila melanogaster

Scientific classification

Kingdom:	Animalia
Phylum:	Arthropoda
Class:	Insecta
Order:	Diptera
Family:	Drosophilidae
Subfamily:	Drosophilinae
Genus:	Drosophila
Subgenus:	Sophophora
Species group:	melanogaster group
Species subgroup:	melanogaster subgroup
Species complex:	melanogaster complex
Species:	D. melanogaster

Binomial name

Drosophila melanogaster
Meigen, 1830^[1]

Drosophila melanogaster (from the Greek for black-bellied dew-lover) is a two-winged insect that belongs to the Diptera, the order of the flies. The species is commonly known as the fruit fly, and is one of the most commonly used model organisms in biology, including studies in genetics, physiology and life history evolution. Flies belonging to the Tephritidae are also called fruit flies, which can lead to confusion.

Physical appearance

Male (left) and female D. melanogaster

Wildtype fruit flies have red eyes, are yellow-brown in colour, and have transverse black rings across their abdomen. They exhibit sexual dimorphism: females are about 2.5 millimetres (0.1 inches) long; males are slightly smaller and the back of their bodies are darker. Males are easily distinguished from females based on colour differences (males have a distinct black patch at the abdomen, less noticeable in recently emerged flies (see fig)) and the sexcombs (a row of dark bristles on the tarsus of the first leg). Furthermore, males have a cluster of spiky hairs (claspers) surrounding the anus and genitals used to attach to the female during mating. There are extensive images at Fly Base.

Life cycle

Egg of D. melanogaster

The developmental period for Drosophila melanogaster varies with temperature, as with many ectothermic species. The shortest development time (egg to adult), 7 days, is achieved at 28 °C.^[2]^[3] Development times increase at higher temperatures (30 °C, 11 days) due to heat stress. Under ideal conditions, the development time at 25 °C is 8.5 days ^[4]^[2]^[3], at 18 °C it takes 19 days^[2]^[3] and at 12 °C it takes over 50 days.^[2]^[3] Under crowded conditions, development time increases ^[5], while the emerging flies are smaller^[5]^[6]. Females lay some 400 eggs (embryos), about five at a time, into rotting fruit or other suitable material such as decaying mushrooms and sap fluxes. The eggs, which are about 0.5 millimetres long, hatch after 12-15 h (at 25 °C).^[2]^[3] The resulting larvae grow for about 4 days (at 25 °C) while molting twice (into 2nd- and 3rd-instar larvae), at about 24 and 48 h after eclosion.^[2]^[3] During this time, they feed on the microorganisms that decompose the fruit, as well as on the sugar of the fruit themselves. Then the larvae encapsulate in the puparium and undergo a four-day-long metamorphosis (at 25 °C), after which the adults eclose (emerge).^[2]^[3]

Females become receptive to courting males at about 8-12 hours after emergence.^[7] Males perform a sequence of five behavioral patterns to court females. First, males orient themselves while playing a courtship song by horizontally extending and vibrating their wings. Soon after, the male positions itself at the rear of the female's adbdomen in a low posture to tap and lick the female genitalia. Finally, the male curls its abdomen, and attempts copulation. Females can reject males by moving away and extruding their ovipositor. The average duration of successful copulation is 30 minutes, during which males transfer a few hundred very long (1.76mm) sperm cells in seminal fluid to the female[1]. Females store the sperm, which may need to compete with other males' stored sperm to fertilize eggs.

The D. melanogaster lifespan is about 30 days at 29 °C.

Model organism in genetics

Drosophila melanogaster types. Eye colors (clockwise): brown, cinnabar, sepia, vermilion, white, wild. Also, the wild-eyed fly has a yellow body, the sepia-eyed fly has an ebony body, and the brown-eyed fly has a black body.

Drosophila melanogaster mutation: yellow cross-veinless forked fruit fly.

A wild fruit fly (left) has antennae, while a fly with the antennapedia mutation (right) has an extra set of feet in the place of antennae.

Drosophila melanogaster is the most studied organism in biological research, particularly in genetics and developmental biology. There are several reasons:

It is small and easy to grow in the laboratory
It has a short generation time (about 2 weeks) and high productivity (females can lay >800 eggs in 1 day)
The mature larvae show giant chromosomes in the salivary glands called polytene chromosomes - "puffs" indicate regions of transcription and hence gene activity.
It has only 4 pairs of chromosomes: 3 autosomal, and 1 sex.
Males do not show meiotic recombination, facilitating genetic studies.
Genetic transformation techniques have been available since 1987.
Its compact genome was sequenced and first published in 2000.^[8]

Charles W. Woodworth is credited with being the first to breed Drosophila in quantity and for suggesting to W. E. Castle that they might be used for genetic research during his time at Harvard University. Beginning in 1910, fruit flies helped Thomas Hunt Morgan accomplish his studies on heredity. "Thomas Hunt Morgan and colleagues extended Mendel's work by describing X-linked inheritance and by showing that genes located on the same chromosome do not show independent assortment. Studies of X-linked traits helped confirm that genes are found on chromosomes, while studies of linked traits led to the first maps showing the locations of genetic loci on chromosomes" (Freman 214). The first maps of Drosophila chromosomes were completed by Alfred Sturtevant.

[edit] Genome

The genome of D. melanogaster (sequenced in 2000, and curated at the FlyBase database^[8]) contains four pairs of chromosomes: an X/Y pair, and three autosomes labeled 2, 3, and 4. The fourth chromosome is so tiny that it is often ignored, aside from its important eyeless gene. Its sequenced genome of ~132 million bases has been annotated^[8] and contains approximately 13,767 protein-coding genes which comprise ~20% of the genome. More than 60% of the genome appears to be functional non-protein-coding DNA^[9] involved in gene expression control. Determination of sex in Drosophila occurs by the ratio of X chromosomes to autosomes, not because of the presence of a Y chromosome as in human sex determination.

Drosophila genes are traditionally named after the phenotype they cause when mutated. For example, the absence of a particular gene in Drosophila will result in a mutant embryo that does not develop a heart. Scientists have thus called this gene tinman, named after the Oz character of the same name (Cf. Azpiazu & Frasch (1993) Genes and Development: 7: 1325-1340.). This system of nomenclature results is a wider range of gene names than in other organisms.

Similarity to humans

About 75% of known human disease genes have a recognizable match in the genetic code of fruit flies (Reiter et al (2001) Genome Research: 11(6):1114-25), and 50% of fly protein sequences have mammalian analogues. An online database called Homophila [2] is available to search for human disease gene homologues in flies and vice versa. Drosophila is being used as a genetic model for several human diseases including the neurodegenerative disorders Parkinson's, Huntington's, and Alzheimer's disease. The fly is also being used to study mechanisms underlying immunity, diabetes, and cancer, as well as drug abuse.

Development and embryogenesis

Main article: Drosophila embryogenesis

Embryogenesis in Drosophila has been extensively studied, as its small size, short generation time, and large brood size makes it ideal for genetic studies. It is also unique among model organisms in that cleavage occurs in a syncytium.

Drosophila melanogaster oogenesis

During oogenesis, cytoplasmic bridges called "ring canals" connect the forming oocyte to nurse cells. Nutrients and developmental control molecules move from the nurse cells into the oocyte. In the figure to the left, the forming oocyte can be seen to be covered by follicular support cells.

After fertilization of the oocyte the early embryo or (syncytial embryo) undergoes rapid DNA replication and 13 nuclear divisions until approximately 5000 to 6000 nuclei accumulate in the unseparated cytoplasm of the embryo. By the end of the 8th division most nuclei have migrated to the surface, surrounding the yolk sac (leaving behind only a few nuclei, which will become the yolk nuclei). After the 10th division the pole cells form at the posterior end of the embryo, segregating the germ line from the syncytium. Finally, after the 13th division cell membranes slowly invaginate, dividing the syncytium into individual somatic cells. Once this process is completed gastrulation starts.

Nuclear division in the early Drosophila embryo happens so quickly there are no proper checkpoints so mistakes may be made in division of the DNA. To get around this problem the nuclei which have made a mistake detach from their centrosomes and fall into the centre of the embryo (yolk sac) which will not form part of the fly.

The gene network (transcriptional and protein interactions) governing the early development of the fruitfly embryo is one of the best understood gene networks to date, especially the patterning along the antero-posterior (AP) and dorso-ventral (DV) axes (See under morphogenesis).

The embryo undergoes well-characterized morphogenetic movements during gastrulation and early development, including germ-band extension, formation of several furrows, ventral invagination of the mesoderm, posterior and anterior invagination of endoderm (gut), as well as extensive body segmentation ^[10] until finally hatching from the surrounding cuticle into a 1st-instar larva.

During larval development, tissues known as imaginal discs grow inside the larva. Imaginal discs develop to form most structures of the adult body, such as the head, legs, wings, thorax and genetalia. Cells of the imaginal disks are set aside during embryogenesis and continue to grow and divide during the larval stages - unlike most other cells of the larva which have differentiated to perform specialized functions and grow without further cell division. At metamorphosis, the larva forms a pupae, inside which the larval tissues are reabsorbed and the imaginal tissues undergo extensive morphogenetic movements to form adult structures.

Behavioral genetics and neuroscience

In 1971, Ron Konopka and Seymour Benzer published "Clock mutants of Drosophila melanogaster", a paper describing the first mutations that affected an animal's behavior. Wild-type flies show an activity rhythm with a frequency of about a day (24 hours). They found mutants with faster and slower rhythms as well as broken rhythms - flies that move and rest in random spurts. Work over the following 30 years has shown that these mutations (and others like them) affect a group of genes and their products that comprise a biochemical or biological clock. This clock is found in a wide range of fly cells, but the clock-bearing cells that control activity are several dozen neurons in the fly's central brain.

Since then, Benzer and others have used behavioral screens to isolate genes involved in vision, olfaction, audition, learning/memory, courtship, pain and other processes, such as longevity.

The first learning and memory mutants (dunce, rutabaga etc) were isolated by William "Chip" Quinn while in Benzer's lab, and were eventually shown to encode components of an intracellular signalling pathway involving cylic AMP, protein kinase A and a transcription factor known as CREB. These molecules were shown to be also involved in synaptic plasticity in Aplysia and mammals.

Male flies sing to the females during courtship using their wing to generate sound, and some of the genetics of sexual behavior have been characterized. In particular, the fruitless gene has several different splice forms, and male flies expressing female splice forms have female-like behavior and vice-versa.

Furthermore, Drosophila has been used in neuropharmacological research, including studies of cocaine and alcohol consumption.

Vision

Stereo images of the fly eye

The compound eye of the fruit fly contains 800 unit eyes or ommatidia, and are one of the most advanced among insects. Each ommatidium contains 8 photoreceptor cells (R1-8), support cells, pigment cells, and a cornea. Wild-type flies have reddish pigment cells, which serve to absorb excess blue light so the fly isn't blinded by ambient light.

Each photoreceptor cell consists of two main sections, the cell body and the rhabdomere. The cell body contains the nucleus while the 100-μm-long rhabdomere is made up of toothbrush-like stacks of membrane called microvilli. Each microvillus is 1-2 μm in length and ~60 nm in diameter.^[11] The membrane of the rhabdomere is packed with about 100 million rhodopsin molecules, the visual protein that absorbs light. The rest of the visual proteins are also tightly packed into the microvillar space, leaving little room for cytoplasm.

The photoreceptors in Drosophila express a variety of rhodopsin isoforms. The R1-R6 photoreceptor cells express Rhodopsin1 (Rh1) which absorbs blue light (480 nm). The R7 and R8 cells express a combination of either Rh3 or Rh4 which absorb UV light (345 nm and 375 nm), and Rh5 or Rh6 which absorb blue (437 nm) and green (508 nm) light respectively. Each rhodopsin molecule consists of an opsin protein covalently linked to a carotenoid chromophore, 11-cis-3-hydroxyretinal. ^[12]

Expression of Rhodopsin1 (Rh1) in photoreceptors R1-R6

As in vertebrate vision, visual transduction in invertebrates occurs via a G protein-coupled pathway. However, in vertebrates the G protein is transducin, while the G protein in invertebrates is Gq (dgq in Drosophila). When rhodopsin (Rh) absorbs a photon of light its chromophore, 11-cis-3-hydroxyretinal, is isomerized to all-trans-3-hydroxyretinal. Rh undergoes a conformational change into its active form, metarhodopsin. Metarhodopsin activates Gq, which in turn activates a phospholipase Cβ (PLCβ) known as NorpA.

PLCβ hydrolyzes phosphatidylinositol (4,5)-bisphosphate (PIP₂), a phospholipid found in the cell membrane, into soluble inositol triphosphate (IP₃) and diacylgycerol (DAG), which stays in the cell membrane. DAG or a derivative of DAG causes a calcium selective ion channel known as TRP (transient receptor potential) to open and calcium and sodium flows into the cell. IP₃ is thought to bind to IP₃ receptors in the subrhabdomeric cisternae, an extension of the endoplasmic reticulum, and cause release of calcium, but this process doesn't seem to be essential for normal vision. ^[13]

Calcium binds to proteins such as calmodulin (CaM) and an eye-specific protein kinase C (PKC) known as InaC. These proteins interact with other proteins and have been shown to be necessary for shut off of the light response. In addition, proteins called arrestins bind metarhodopsin and prevent it from activating more Gq.

A potassium-dependent sodium/calcium exchanger known as NCKX30C pumps the calcium out of the cell. It uses the inward sodium gradient and the outward potassium gradient to extrude calcium at a stoichiometry of 4 Na⁺/ 1 Ca⁺⁺, 1 K⁺.^{[citation needed]}

TRP, InaC, and PLC form a signaling complex by binding a scaffolding protein called InaD. InaD contains five binding domains called PDZ domains which specifically bind the C termini of target proteins. Disruption of the complex by mutations in either the PDZ domains or the target proteins reduces the efficiency of signaling. For example, disruption of the interaction between InaC, the protein kinase C, and InaD results in a delay in inactivation of the light response.

Unlike vertebrate metarhodopsin, invertebrate metarhodopsin can be converted back into rhodopsin by absorbing a photon of orange light (580 nm).

Approximately two-thirds of the Drosophila brain (about 200,000 neurons total) is dedicated to visual processing. Although the spatial resolution of their vision is significantly worse than that of humans, their temporal resolution is approximately ten times better.

Flight

The wings of a fly are capable of beating at up to 220 times per second. Flies fly via straight sequences of movement interspersed by rapid turns called saccades. During these turns, a fly is able to rotate 90 degrees in fewer than 50 milliseconds.

Drosophila, and probably many other flies, have optic nerves which lead directly to the wing muscles (while in other insects they always lead to the brain first), making it possible for them to react extremely quickly.^{[citation needed]}

It was long thought that the characteristics of Drosophila flight were dominated by the viscosity of the air, rather than the inertia of the fly body. However, recent research by Michael Dickinson and Rosalyn Sayaman has indicated that flies perform banked turns, where the fly accelerates, slows down while turning, and accelerates again at the end of the turn. This indicates that inertia is the dominant force, as is the case with larger flying animals.

Courtship and mating

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When two Drosophila melanogaster of the opposite sex encounter one another, they often first exhibit "cleaning behavior". After this behavior is engaged for some time, the male will proceed towards the rear of the female from either the left or right side. He will then begin a "dance" in which he describes a semi-circle around the female. During this dance the male may or may not vibrate his wings. If the female is not receptive, she will move away and courtship ends. If she is receptive however, the male will approach her rear and make contact with her using his proboscis. The female may then scissor her wings and allow the male to mount. When intercourse, which usually takes about 30 minutes, has finished, the female will dislodge the male with a violent kicking of her hind legs.

Go fishing, solved developmental clues: Zebrafish

2007-06-07T07:31:00.000Z

Danio rerio

Scientific classification

Kingdom:	Animalia
Phylum:	Chordata
Class:	Actinopterygii
Order:	Cypriniformes
Family:	Cyprinidae
Genus:	Danio
Species:	D. rerio

Binomial name

Danio rerio
(Hamilton-Buchanan, 1822)

The zebrafish (Danio rerio) is a tropical fish belonging to the minnow family (Cyprinidae).^[1] It is a popular aquarium fish, where it is frequently sold under the trade name zebra danio, and is also an important model organism.

Characteristics

The zebrafish arose in the Ganges region in Eastern India and is also native to Pakistan, Bangladesh, Nepal and Myanmar. It commonly inhabits streams, canals, ditches, ponds and slow-moving to stagnant water bodies, including rice fields.

The fish is named for its five uniform, pigmented, horizontal blue stripes on the side of the body, all of which extend to the end of the caudal fin. Males are torpedo shaped and have gold stripes between the blue stripes; females have a larger, whitish belly and have silver stripes instead of gold. The zebrafish grows to 3.8 cm, lives for around 5 years, and produces 300-500 eggs per spawning.

Recently, transgenic zebrafish have become commercially available that express green fluorescent protein,red fluorescent protein, and yellow fluorescent protein. These fish are tradenamed GloFish. Other varieties include leopard and longfin.

Varieties

The Leopard danio, previously known as Danio frankeri, is a spotted colour morph of the zebrafish Danio rerio caused by a pigment mutation.^[2] Xanthistic forms of both the zebra and leopard pattern, along with long-finned varieties have been obtained via selective breeding programs for the aquarium trade.^[3]

Aquarium Care

Zebrafish are hardy fish and considered good for beginner aquarists. Their ease of keeping and breeding, beauty, price and broad availability may all contribute to their popularity. However, they are susceptible to Oodinium, or Velvet disease, Microsporidia (Pseudoloma neurophilia),and mycobacterium species.

Zebrafish as an introduced species

Zebrafish have been introduced in Japan and the United States but they have not established in either location ^[1]. They have also been sighted in Colombia, presumably having escaped from an aquarium.

Model organism for development and genetics

Zebrafish chromatophores, shown here mediating background adaptation, are studied by scientists

D. rerio are a common and useful model organism for studies of vertebrate development and gene function. They may supplement or replace higher vertebrate models, such as rats and mice. Pioneering work of George Streisinger at the University of Oregon established the zebrafish as a model organism; its importance was consolidated by large scale forward genetic screens (commonly referred to as the Tübingen/Boston screens). The scholarly journal Development devoted an issue to research using the fish in celebration of this landmark. ^[2] D. rerio is one of the few species of fish to have been flown into space (see Animals in space).

Zebrafish embryonic development provides advantages over other vertebrate model organisms. Although the overall generation time of zebrafish is comparable to that of mice, zebrafish embryos develop rapidly, progressing from eggs to larvae in under three days. The embryos are large, robust, and transparent and develop externally to the mother, characteristics which all facilitate experimental manipulation and observation. Their nearly constant size during early development facilitates simple staining techniques, and drugs may be administered by adding directly to the tank. Unfertilised eggs can be made to divide, and the two-celled embryo fused into a single cell, creating a fully homozygous embyo.

A common reverse genetics technique is to reduce gene expression or modify splicing in zebrafish using Morpholino antisense technology. Morpholino oligonucleotides are stable, synthetic macromolecules that contain the same bases as DNA or RNA; by binding to complementary RNA sequences, they reduce the expression of specific genes. The journal Genesis devoted an issue to research using Morpholino oligos^[3], mostly in D. rerio. Morpholino oligonucleotides can be injected into one cell of a zebrafish embryo after the 32-cell stage, producing an organism in which gene expression is reduced in only the cells descended from the injected cell. However, cells in the early embryo (<32>[4], [5], allowing diffusion of Morpholinos between cells. A known problem with gene knockdowns in zebrafish is that, because the genome underwent a duplication after the divergence of ray-finned fishes and lobe-finned fishes, it is not always easy to silence the activity one of the two gene paralogs reliably due to complementation by the other paralog.

Despite the complications of the zebrafish genome a number of attempts have also been made to develop global platforms for analysis of both gene expression by microarrays and also promoter regulation using ChIP-on-chip.

Zebrafish have the ability to regenerate fins, skin, the heart and the brain (in larval stages). Zebrafish have also been found to regenerate photoreceptors and retinal neurons following injury. The mechanisms of this regeneration are unknown, but are currently being studied. Researchers frequently cut the dorsal and ventral tail fins and analyze their regrowth to test for mutations. This research is leading the scientific community in the understanding of healing/repair mechanisms in vertebrates.

The results of genetic engineering in these fishes have been used to produce the Glofish, an aquarium pet with fluorescent pigments. Other varieties include golden, sandy and long fin fish.

In December 2005, a study of the golden strain identified the gene responsible for the unusual pigmentation of this strain as SLC24A5, a solute carrier that appears to be required for melanin production, and confirmed its function with a Morpholino knockdown. The orthologous gene was then characterized in humans and a one base pair difference was found to segregate strongly between fair skinned Europeans and dark skinned Africans. ^[6] This study featured on the cover of the academic journal Science and demonstrates the power of zebrafish as a model organism in the relatively new field of comparative genomics.

In January 2007, Chinese researchers at Fudan University raised genetically modified fish that can detect estrogen pollution in lakes and rivers, showing environmental officials what waterways need to be treated for the substance, which is linked to male infertility. Song Houyan and Zhong Tao, two professors at Fudan's molecular medicine lab, spent three years cloning estrogen-sensitive genes and injecting them into the fertile eggs of zebrafish. The modified fish turn green if they are placed into water that is polluted by estrogen^[7].

Small Wonder : Caenorhabditis elegans

2007-06-07T07:25:00.000Z

Caenorhabditis elegans

An adult hermaphrodite C. elegans worm

Scientific classification

Kingdom:	Animalia
Phylum:	Nematoda
Class:	Secernentea
Order:	Rhabditida
Family:	Rhabditidae
Genus:	Caenorhabditis
Species:	elegans

Binomial name

Caenorhabditis elegans
Maupas, 19

Caenorhabditis elegans
is a free-living nematode (roundworm), about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner ^[1] and it has since been used extensively as a model organism.

Biology

C. elegans is unsegmented, vermiform, bilaterally symmetrical, with a cuticle integument, four main epidermal cords and a fluid-filled pseudocoelomate cavity. Members of the species have many of the same organ systems as other animals. In the wild, they feed on bacteria that develop on decaying vegetal matter. C. elegans has both a hermaphrodite sex, and a very rare male population, which makes up 0.05% of the total C. elegans on average. The basic anatomy of C. elegans includues a mouth, pharynx, intestine, gonad, and collagenous cuticle. Males have a single-lobed gonad, vas deferens, and a tail specialized for mating. Hermaphrodites have two ovaries, oviducts, spermatheca, and a single uterus.

C. elegans eggs are laid by the hermaphrodite. After hatching, they pass through four larval stages (L1-L4). When crowded or in the absence of food, C. elegans can enter an alternative third larval stage called the dauer state. Dauer larvae are stress-resistant and do not age. Hermaphrodites produce sperm during the L4 stage, and lay eggs as adults ^[2]. The male can inseminate the hermaphrodite, which will use male sperm preferentially. At 20°C, the laboratory strain of C. elegans has an average life span of approximately 2-3 weeks and a generation time of approximately 4 days. Hermaphrodites can mate with males or self-fertilize.

C. elegans has five pairs of autosomes and one pair of sex chromosomes. Sex in C. elegans is based on an X0 sex-determination system. Hermaphrodite C. elegans have a matched pair of sex chromosomes (XX); the rare males have only one sex chromosome (X0).

C. elegans as a model organism

C. elegans is used as a model organism for a variety of reasons, including economy, and the ease of maintaining a population in the laboratory. Worms can be frozen and subsequently thawed and remain viable, thus ensuring easy long-term storage of different worm strains. Because the complete cell lineage of the species has been determined, C. elegans has proven especially useful for studying cellular differentiation.

From a research perspective, C. elegans has the advantage of being a multicellular eukaryotic organism which is simple enough to be studied in great detail. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out. These patterns of cell lineage are largely invariant between individuals, in contrast to mammals where cell development from the embryo is more largely dependent on cellular cues. In both sexes, a large number of additional cells (131 in the hermaphrodite, most of which would otherwise become neurons), are eliminated by programmed cell death (apoptosis).

Wild-type C. elegans hermaphrodite stained to highlight the nuclei of all cells

In addition, C. elegans is one of the simplest organisms with a nervous system. In the hermaphrodite, this comprises 302 neurons whose pattern of connectivity has been completely mapped out, and shown to be a small-world network ^[3]. Research has explored the neural mechanisms responsible for several of the more interesting behaviors shown by C. elegans, including chemotaxis, thermotaxis, mechanotransduction, and male mating behavior. Interestingly, the neurons fire no action potentials.

A useful feature of C. elegans is that it is relatively straightforward to disrupt the function of specific genes by RNA interference (RNAi). Silencing the function a gene in this way can sometimes allow a researcher to infer what the function of that gene may be. The nematode can either be soaked in (or injected with) a solution of double stranded RNA, the sequence of which is complentary to the sequence of the gene that the researcher wishes to disable. Alternatively, worms can be fed on genetically transformed bacteria which express the double stranded RNA of interest.

C. elegans has also been useful in the study of meiosis. As sperm and egg nuclei move down the length of the gonad, they undergo a temporal progression through meiotic events. This progression means that every nuclei at a given position in the gonad will be at roughly the same step in meiosis, eliminating the difficulties of heterogeneous populations of cells.

The organism has also been identified as a model for nicotine dependence as it has been found to experience the same symptoms humans experience when they quit smoking. ^[4]

As for most model organisms, there is a dedicated online database for the species that is actively curated by scientists working in this field. The WormBase database attempts to collate all published information on C. elegans and other related nematodes.

The Genome

C. elegans was the first multicellular organism to have its genome completely sequenced. The finished genome sequence was published in 1998,^[5] although a number of small gaps were present (the last gap was finished by October 2002). The C. elegans genome sequence is approximately 100 million base pairs long and contains approximately 20,000 genes. The vast majority of these genes encode for proteins but there are likely to be as many as 1,000 RNA genes. Scientific curators continue to appraise the set of known genes, such that new gene predictions continue to be added and incorrect ones modified or removed.

In 2003, the genome sequence of the related nematode C. briggsae was also determined, allowing researchers to study the comparative genomics of these two organisms ^[6]. Work is now ongoing to determine the genome sequences of more nematodes from the same genus such as C. remanei [1], C. japonica [2] and C. brenneri [3]. These newer genome sequences are being determined by using the whole genome shotgun technique which means that the resulting genome sequences are likely to not be as complete or accurate as C. elegans (which was sequenced using the 'hierarchical' or clone-by-clone appoach).

The official version of the C. elegans genome sequence continues to change as and when new evidence reveals errors in the original sequencing (DNA sequencing is not an error free process). Most changes are usually minor, adding or removing only a few base pairs (bp) of DNA. E.g. the WS169 release of WormBase (December 2006) lists a net gain of 6 bp to the genome sequence ^[7]. Occasionally more extensive changes are made, e.g. the WS159 release of May 2006 added over 300 bp to the sequence ^[8].

Nematode evolution

It has been shown that a small number of conserved protein sequences from sponges are more similar to humans than to C. elegans ^[9]. This suggests that there has been an accelerated rate of evolution in the C. elegans lineage. The same study found that several phylogenetically ancient genes are not present in C. elegans.

C. elegans scientists

In 2002, the Nobel Prize for Medicine was awarded to Sydney Brenner, H. Robert Horvitz and John Sulston for their work on the genetics of organ development and programmed cell death (PCD) in C. elegans. The 2006 Nobel Prize in Physiology or Medicine was awarded to Andrew Fire and Craig C. Mello, for their discovery of RNA interference in C. elegans.^[10]

Because all research into C. elegans essentially started with Sydney Brenner in the 1970's, many scientists working in this field share a close connection to Brenner (they either worked as a post-doctoral or post-graduate researcher in Brenner's lab or in the lab of someone who previously worked with Brenner). Because most people who worked in his lab went on to establish their own worm research labs, there is now a fairly well documented 'lineage' of C. elegans scientists. This lineage was recorded in some detail at the 2003 International Worm Meeting and the results were stored in the Wormbase database.

The most impt factors : Transcription factor

2007-06-06T20:21:00.000Z

In molecular biology, a transcription factor is a protein that works in concert with other proteins to either promote or suppress the transcription of genes.^[1]^[2] Hence transcription factors control when and where genes (and the proteins encoded by those genes) are expressed.

More specifically, transcription factors regulate the binding of RNA polymerase to DNA and as a result control the subsequent transcription of DNA into messenger RNA and eventually protein. Transcription factors bind to specific sequences of DNA upstream or downstream to the gene they regulate and then either enhance or repress transcription of these genes by assisting or blocking RNA polymerase binding respectively. A cluster of transcription factors is the preinitiation complex (PIC) that recruits and activates RNA polymerase. Conversely, repressor transcription factors inhibit transcription by blocking the attachment of activator proteins.

A defining characteristic of transcription factors is that they contain a DNA binding domain (DBD) which bind to gene specific regulatory sites (e.g., promoter sequences). In addition, transcription factors often contain a second domain that sense external signals and in response transmit these signals to the rest of the transcription complex resulting in up or down regulation of gene expression. In some cases the DBD and signal sensing domains reside on separate proteins that associate within the transcription complex to regulate gene expression.

Additional proteins such as coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, while also playing crucial roles in gene regulation, lack DNA binding domains, and therefore are not classified as transcription factors.

Regulation

The regulation of transcription is a highly complex process as it is dependent upon a number of factors including which transcription factors and other coregulatory proteins are present within a particular cell as well as the local 3-dimensional structure of the gene (chromatin).

Initial models, based on in vitro experiments suggested that the assembly of transcription factors dictated by the DNA sequence. It is, however, becoming increasingly obvious that the events leading to activation of transcription are dependent on a large number of factors and are highly intertwined. Furthermore epigenetic information present on DNA appears to play an important role in transcriptional activation.

Classes

There are three classes of transcription factors:

General transcription factors are involved in the formation of a preinitiation complex. The most common are abbreviated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH. They are ubiquitous and interact with the core promoter region surrounding the transcription start site(s) of all class II genes.
Upstream transcription factors are proteins that bind somewhere upstream of the initiation site to stimulate or repress transcription.
Inducible transcription factors are similar to upstream transcription factors but require activation or inhibition.

General Definition- Protein involved in recognition by RNA polymerases of specific regulatory sequences in eukaryotic genes.

Structural Binding Motifs

DNA-Binding motifs

Basic-helix-loop-helix (bHLH) bind DNA with two alpha helices containing basic amino acid residues which are linked by a loop and are typically dimeric.
Helix-turn-helix (HTH) bind the major groove of the DNA.
Zinc fingers function as structural platforms for DNA binding.
The MADS-box encodes a DNA-binding domain found in e.g. floral homeotic genes.

Protein-Binding motifs

Leucine zippers function in associating the transcription factors with each other.

Transcription Factor-Binding motifs found in DNA

G-quadruplex Motifs are a focus for current studies in their role as a TF binding site.

Examples of transcription factors

STAT

The Signal Transducers and Activator of Transcription (STAT) proteins regulate many aspects of cell growth, survival and differentiation. The transcription factors of this family are activated by the Janus Kinase JAK and dysregulation of this pathway is frequently observed in primary tumors and leads to increased angiogenesis and enhanced survival of tumors. Knockout studies have provided evidence that STAT proteins are involved in the development and function of the immune system and play a role in maintaining immune tolerance and tumor surveillance.

Function of STAT proteins

STAT proteins were originally described as latent cytoplasmic transcription factors that require phosphorylation for nuclear retention. The unphosphorylated STAT proteins shuttle between the cytosol and the nucleus waiting for its activation signal. Once the activated transcription factors reach the nucleus, they bind to a consensus DNA-recognition motif called gamma activated sites (GAS) in the promoter region of cytokine-inducible genes and activate transcription of these genes.....

Activation of STAT proteins

Extracellular binding of Cytokines induces activation of the intracellular Janus kinase that phosphorylates a specific tyrosine residue in the STAT protein which promotes the dimerization of STAT monomers via their SH2 domain. The phosphorylated dimer is then actively transported in the nucleus via importin a/b and RanGDP complex. Once inside the nucleus the active STAT dimer binds to cytokine inducible promoter regions of genes containing gamma activated site (GAS) motif and activate transcription of this proteins. The STAT protein can be dephosphorylated by nuclear phosphatases which leads to inactivation of STAT and the transcription factor becomes transported out of the nucleus by exportin crm1/RanGTP.

Its all @ the ENDS : Telomere

2007-06-06T20:17:00.000Z

A telomere is a region of highly repetitive DNA at the end of a linear chromosome that functions as a disposable buffer. Every time linear eukaryotic chromosomes are replicated during late S phase, the DNA polymerase complex is incapable of replicating all the way to the end of the chromosome; if it were not for telomeres, this would quickly result in the loss of vital genetic information, which is needed to sustain a cell's activities. Every time a cell with linear genes divides, it will lose a small piece of one of its strands of DNA. This process has been referred to by James Watson and Alexei Olovnikov as the "end replication problem" (1971). It is believed that telomeres have a function in the ageing process.

Nature and function of telomeres

The telomere is a "ribonucleoprotein complex" composed of a protein component and an RNA primer sequence which acts to protect the terminal ends of chromosomes. This prevents chromosomal fraying and prevents the ends of the chromosome from being processed as a double strand DNA break, which could lead to chromosome-to-chromosome telomere fusions. Telomeres are extended by telomerases, part of a protein subgroup of specialized reverse transcriptase enzymes known as TERT (TElomerase Reverse Transcriptases) that are involved in synthesis of telomeres in humans and many other, but not all, organisms. However, because of DNA replication mechanisms and because TERT expression is repressed in many types of human cells, the telomeres of these cells shrink a little bit every time a cell divides although in other cellular compartments which require extensive cell division, such as stem cells and certain white blood cells, TERT is expressed and telomere length is maintained.

Structure of parallel quadruplexes that can be formed by human telomeric DNA. Image created from NDB UD0017.

In addition to its TERT protein component, telomerase also contains a piece of template RNA known as the TERC (TElomerase RNA Component) or TR (Telomerase RNA). In humans, this TERC telomere sequence is a repeating string of TTAGGG, between 3 and 20 kilobases in length. There are an additional 100-300 kilobases of telomere-associated repeats between the telomere and the rest of the chromosome. Telomere sequences vary from species to species, but are generally GC-rich. These GC-rich sequences can form four-stranded structures (G-quadruplexes), with sets of four bases held in plane and then stacked on top of each other with either a sodium or potassium ion between the planar quadruplexes.

In most prokaryotes, chromosomes are circular and thus do not have ends to suffer premature replication termination. A small fraction of bacterial chromosomes (such as those in Streptomyces and Borrelia) are linear and possess telomeres, which are very different from those of the eukaryotic chromosomes in structure and functions.

In most multicellular eukaryotes, telomerase is only active in germ cells. There are theories that the steady shortening of telomeres with each replication in somatic (body) cells may have a role in senescence and in the prevention of cancer. This is because the telomeres act as a sort of time-delay "fuse", eventually running out after a certain number of cell divisions and resulting in the eventual loss of vital genetic information from the cell's chromosome with future divisions.

If telomeres become too short, they will potentially unfold from their presumed closed structure. It is thought that the cell detects this uncapping as DNA damage and will enter cellular senescence, growth arrest or apoptosis depending on the cell's genetic background (p53 status). Uncapped telomeres also result in chromosomal fusions. Since this damage cannot be repaired in normal somatic cells, the cell may even go into apoptosis. Many aging-related diseases are linked to shortened telomeres. Organs deteriorate as more and more of their cells die off or enter cellular senescence.

At the very distal end of the telomere is a 300 bp single-stranded portion which forms the T-Loop. This loop is analogous to a 'knot' which stabilizes the telomere; preventing the telomere ends from being recognized as break points by the DNA repair machinery. Should non-homologous end joining occur at the telomeric ends, chromosomal fusion will result. The T-loop is held together by seven known proteins; most notably TRF1, TRF2, POT1, TIN1, and TIN2.

A study published in the May 3, 2005 issue of the American Heart Association journal Circulation found that weight gain and increased insulin resistance were correlated with greater telomere shortening over time.

Telomere shortening

Lagging strand during DNA replication

"Telomeres" shorten because of the end replication problem that is exhibited during DNA replication in eukaryotes only. Because DNA replication does not begin at either end of the DNA strand, but starts in the center, and considering that all DNA polymerases that have been discovered move in the 5' to 3' direction, one finds a leading and a lagging strand on the DNA molecule being replicated.

On the leading strand, DNA polymerase can make a complementary DNA strand without any difficulty because it goes from 5' to 3'. However, there is a problem going in the other direction on the lagging strand. To counter this, short sequences of RNA acting as primers attach to the lagging strand a little way ahead of where the initiation site was. The DNA polymerase can start replication at that point and go to the end of the initiation site. This causes the formation of Okazaki fragments. More RNA primers attach further on the DNA strand and DNA polymerase comes along and continues to make a new DNA strand.

Eventually, the last RNA attaches, and DNA polymerase and DNA ligase come along to convert the RNA (of the primers) to DNA, and seal the gaps in between the Okazaki fragments. But in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer. This happens at all the sites of the lagging strand, but it doesn't happen at the end where the last RNA primer is attached. Ultimately, that RNA is destroyed by enzymes that degrade RNA left on the DNA. Thus, a section of telomeres is lost during each cycle of replication at the 5' end of both the leading and lagging strands.

Extending telomeres

The phenomenon of limited cellular division was first observed by Leonard Hayflick. Significant discoveries were made by the team led by Professor Elizabeth Blackburn at the University of California - San Francisco. In 1998, Geron Corp. developed techniques for extending telomeres, and demonstrated that this prevented cellular senescence.^{[citation needed]}

Advocates of human life extension promote the idea of lengthening the telomeres in certain cells through temporary activation of telomerase (by drugs), or possibly permanently by gene therapy. They reason that this would extend human life. So far these ideas have not been proven in humans.

However, it has been hypothesized that there is a trade-off between cancerous tumor suppression and tissue repair capacity, and that by lengthening telomeres we might slow aging and in exchange increase vulnerability to cancer (Weinstein and Ciszek, 2002).

A study done with the nematode worm species Caenorhabditis elegans indicates that there is a correlation between lengthening telomeres and a longer lifespan. Two groups of worms were studied which differed in the amount of the protein HRP-1 their cells produced and, resulting in telomere lengthening in the mutant worms. The worms with the longer telomeres lived 24 days on average, about 20 percent longer than the normal worms. A side effect of the mutation was an increased resistance to the effects of heat exposure. The reasons for that effect are unclear. (Joeng et al., 2004).

Techniques to extend telomeres could be useful for tissue engineering, because they might permit healthy, noncancerous mammalian cells to be cultured in amounts large enough to be engineering materials for biomedical repairs.

However, there are several issues that still need to be cleared up. First, it is not even certain whether the relationship between telomeres and aging is causal. Although this is indeed probably so because changing telomere lengths are usually associated with changing speed of senescence, the relationship may well be the other way around, with telomere shortening a consequence of and not a reason for aging. That the role of telomeres is far from being understood is demonstrated by two recent studies on long-lived seabirds:

In 2003, scientists observed that the telomeres of Leach's Storm-petrel (Oceanodroma leucorhoa) seem to lengthen with chronological age, the first observed instance of such behaviour of telomeres[1]. In 2006, Juola et al. reported that in another, unrelated long-lived seabird species, the Great Frigatebird (Fregata minor), telomere length did decrease until at least c.40 years of age (i.e. probably over the entire lifespan), but the speed of decrease slowed down massively with increasing ages, and that rates of telomere length decrease varied strongly between individual birds. They concluded that in this species (and probably in frigatebirds and their relatives in general), telomere length could not be used to determine a bird's age sufficiently well. Thus, it seems that there is much more variation in the behavior of telomere length than initially believed, and more research into the topic is clearly warranted before any firm conclusions can be drawn or even practical applications tested.

Telomere Length Assay

Several techniques are currently employed to assess average telomere length in eukaryotic cells. The most widely used method is the Terminal Restriction Fragment (TRF) southern blot which involves hybridization of a radioactive 32P-(TTAGGG)n oligonucleotide probe to Hinf / Rsa I digested genomic DNA embedded on a nylon membrane; and subsequently exposed to autoradiographic film or phosphoimager screen. Another histochemical method involves fluorescent in situ hybridization (FISH). These methods however, require significant amounts of genomic DNA (2-20 micrograms) and labor which renders its use limited in large epidemiological studies. These impediments have been overcome with a novel Real-Time PCR assay for telomere length developed by Richard Cawthon at the University of Utah. This assay involves determining the Telomere-to-Single Copy Gene (T/S)ratio which is demonstrated to be proportional to the average telomere length in a cell (Cawthon 2002). The Real-Time PCR assay has been since redeveloped in a high-throughput 384-well format for use with an Applied Biosystems 7900HT by Jason Wong of the Brigham and Women's Hospital / Harvard Medical School; making the assay feasible for use in large cohort studies.

Telomere sequences

Some known telomere sequences
Group	Organism	Telomeric repeat (5' to 3' toward the end)
Vertebrates	Human, mouse, Xenopus	TTAGGG
Filamentous fungi	Neurospora crassa	TTAGGG
Slime moulds	Physarum, Didymium	TTAGGG
Slime moulds	Dictyostelium	AG(1-8)
Kinetoplastid protozoa	Trypanosoma, Crithidia	TTAGGG
Ciliate protozoa	Tetrahymena, Glaucoma	TTGGGG
	Paramecium	TTGGG(T/G)
	Oxytricha, Stylonychia, Euplotes	TTTTGGGG
Apicomplexan protozoa	Plasmodium	TTAGGG(T/C)
Higher plants	Arabidopsis thaliana	TTTAGGG
Green algae	Chlamydomonas	TTTTAGGG
Insects	Bombyx mori	TTAGG
Roundworms	Ascaris lumbricoides	TTAGGC
Fission yeasts	Schizosaccharomyces pombe	TTAC(A)(C)G(1-8)
Budding yeasts	Saccharomyces cerevisiae	TGTGGGTGTGGTG (from RNA template) or G(2-3)(TG)(1-6)T (consensus)
	Candida glabrata	GGGGTCTGGGTGCTG
	Candida albicans	GGTGTACGGATGTCTAACTTCTT
	Candida tropicalis	GGTGTA[C/A]GGATGTCACGATCATT
	Candida maltosa	GGTGTACGGATGCAGACTCGCTT
	Candida guillermondii	GGTGTAC
	Candida pseudotropicalis	GGTGTACGGATTTGATTAGTTATGT
	Kluyveromyces lactis	GGTGTACGGATTTGATTAGGTATGT

Telomeres and cancer

Telomere maintenance activity is a hallmark in approximately 90% of cancers in almost all mammalian organisms. In humans, cancerous tumors acquire indefinite replicative capacity by over-expressing telomerase. However, a sizeable fraction of cancerous cells employ alternative lengthening of telomeres (ALT), a non-conservative telomere lengthening pathway involving the transfer of telomere tandem repeats between sister-chromatids. The mechanism by which ALT is activated is not fully understood because these exchange events are difficult to assess in vivo.

Ah ! Mingling With Interactions: Two-hybrid

2007-06-06T20:12:00.000Z

Overview of two-hybrid assay as follows. Binding and activating domains of transcription factor GAL4 are split and fused to two proteins being tested for interactions. If the two proteins interact, transcription of "reporter gene" occurs. Otherwise, "reporter gene" is silent.

Two-hybrid screening is a molecular biology technique used to discover protein-protein interactions^[1] and protein-DNA interactions^[2]^[3] by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For the purposes of two-hybrid screening, the transcription factor is split into two separate fragments, called the binding domain (BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for activation of transcription.^[1]^[2]

History

Pioneered by Fields and Song in 1989, the technique was originally designed to detect protein-protein interactions using the GAL4 transcriptional activator of the yeast Saccharomyces cerevisiae. The GAL4 protein activated transcription of a protein involved in galactose utilization which formed the basis of selection.^[4] Since then, the same principle has been adapted to describe many alternative methods including some that detect protein-DNA interactions and use Escherichia coli instead of yeast.^[3]

Basic Premise

Key to the two-hybrid screen, is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in close proximity to each other without direct binding.^[5] This means that even though the transcription factor is split into two fragments, it can still activate transcription when the two fragments are indirectly connected.

The most common screening approach is the yeast two-hybrid assay^[6]. This system often utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lacks these nutrients, the yeast fail to survive. This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids. In yeast two-hybrid screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain.

Plasmids are engineered to produce a protein product in which the DNA-binding domain (BD) fragment is fused onto a protein while another plasmid is engineered to produce a protein product in which the activation domain (AD) fragment is fused onto another protein. The protein fused to the BD may be referred to as the bait protein and is typically a known protein that the investigator is using to identify new binding partners. The protein fused to the AD may be referred to as the prey protein and can be either a single known protein or a library of known or unknown proteins. In this context, a library may consist of a collection of protein-encoding sequences that represent all the proteins expressed in a particular organism or tissue or may be generated by synthesising random DNA sequences.^[3] Regardless of the source, they are subsequently incorporated into the protein-encoding sequence of a plasmid which are then transfected into the cells chosen for the screening method.^[3] This technique, when using a library, assumes that each cell is transfected with no more than a single plasmid and that therefore, each cell ultimately expresses no more than a single member from the (or each, if two libraries are selected against one another) protein library.

If the bait and prey proteins interact (i.e. bind), then the AD and BD of the transcription factor are indirectly connected, bringing the AD in proximity to the transcription start site and transcription of reporter gene(s) can occur. If the two proteins do not interact, there is no transcription of the reporter gene. In this way, a successful interaction between the fused protein is linked to a change in the cell phenotype.^[1]

The challenge of separating those cells which express proteins which happen to interact with their counterpart fusion proteins, from those which do not, is addressed in the following section, 'Reporter and selection genes'.

Reporter and selection genes

In order to link the interaction to a change in observable phenotype, a reporter gene is provided with the upstream activation sequence (UAS) to which the binding domain binds, resulting in gene expression in successful cases of interaction. Since its inception in 1989, the technique has been combined with a number of different reporter genes which can allow selection through a simple colour change or through automatic death of cells in which the interaction does or does not take place.^[1]

The lacZ reporter gene will allow the highlighting of cells in which the UAS-BD-AD interaction is taking place.^[1] β-galactosidase, the gene product of lacZ produces a blue colouration through the metabolism of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) which allows the experimenter to manually choose the individuals which host proteins displaying the required [level of] interaction.^[2]

Manual differentiation of cells according to colour may be acceptable for small numbers of cells, as when investigating a small number of proteins, but when a large library of proteins must be screened, some automation is necessary.^[2] This automation is provided by a number of genes and gene systems which either cause the death of cells that aren't hosting interactions or vice versa, leaving only the cells expressing the proteins of interest.^[2]

For more autonomy, alternative reporter genes exist that enable automatic deletion of individuals that fail to express the reporter or vice versa.

Some projects utilise a dual reporter system in which two reporter genes are used in order to help substantiate the interaction.^[3]^[1] When developing their HIS3 selection system, Joung et al. (2000) included an aadA gene, downstream of the HIS3 gene such that it may be expressed at the same time as the HIS3 gene. The aadA gene confers spectinomycin resistance and although insufficient as a selection gene in itself, was used to maintain selection pressure and provide greater stringency.

[edit] Positive selection

In positive selection, the individuals that host a successful interaction are able to live on the provided media, while those failing to host this interaction die. This can be achieved by combining a media that is lacking an essential nutrient with a strain of an organism which is dependent on expression of the reporter gene in order to produce this nutrient. Examples include the HIS3 gene, encoding a protein required for histidine synthesis, the LEU2 gene, encoding a protein required for leucine synthesis and the URA3 gene, encoding a protein required for uracil synthesis.^[1]

The positive selection method is used in investigations in which the investigators are aiming to discover proteins which interact. Applications include the discovery of homologues in other species and the discovery of members of a protein family (containing a particular domain and therefore binding to a compatible domain).^[1] This HIS3 gene for example can be use with E. coli cells bearing a deletion in their homologous hisB gene (ΔhisB cells).^[2]

Counter-selection

Alternatively, reporter genes conferring sensitivity to an agent supplied in the media, will kill cells expressing the reporter, thereby removing cells that host an interaction. Examples of genes used in the counter selection method include CYH2 (confers sensitivity to cycloheximide), CAN1 and URA3.^[1] Alternatively, using repressor domains or other negative regulators in place of activation domains, counter-selection may be achieved using the same reporter genes used in positive selection methods. One such negative regulator is the yeast GAL80 domain which binds and inactivates the transcriptional activator region of GAL4.^[1]

Such counter-selection methods may be used in investigations aiming to discover a mutation, chemical or protein that will interfere with the interaction.

Fixed domains

In any study, some of the protein domains, those under investigation, will be varied according to the goals of the study whereas other domains, those that are not themselves being investigated, will be kept constant. For example in a two-hybrid study to select DNA-binding domains, the DNA-binding domain, BD, will be varied whilst the two interacting proteins, the bait and prey, will need to be kept constant in order to maintain a strong binding between the BD and AD. There are a number of domains from which to choose the BD, bait and prey and AD, if these are to remain constant. In protein-protein interaction investigations, the BD may be chosen from any of many strong DNA-binding domains such as Zif268.^[2] A frequent choice of bait and prey domains are residues 263-352 of yeast Gal11P with a N342V mutation^[2] and residues 58-97 of yeast Gal4,^[2] respectively. These domains can be used in both yeast- and bacterial-based selection techniques and are known to bind together strongly.^[1]^[2]

The AD chosen must be able to activate transcription of the reporter gene, using the cell's own transcription machinery. Thus, the variety of ADs available for use in yeast-based techniques may not be suited to use in their bacterial-based analogues. The herpes simplex virus-derived AD, VP16 and yeast Gal4 AD have been used with success in yeast^[1] whilst a portion of the α-subunit of E. coli' RNA polymerase has been utilised in E. coli-based methods.^[2]^[3]

Whilst powerfully-activating domains may allow greater sensitivity towards weaker interactions, conversely, a weaker AD may provide greater stringency.

Construction of expression plasmids

There are a number of engineered genetic sequences which must be incorporated into the host cell in order to perform a two-hybrid analysis or one of its derivative techniques. The considerations and methods used in the construction and delivery of these sequences differ according to the needs of the assay and the organism chosen as the experimental background.

There are two broad categories of hybrid library: random libraries and cDNA-based libraries. A cDNA library is constituted by the cDNA produced through reverse transcription of mRNA collected from specific cells of types of cell. This library can be ligated into a construct so that it is attached to the BD or AD being used in the assay.^[1] A random library uses lengths of DNA of random sequence in place of these cDNA sections. A number of methods exist for the production of these random sequences, including cassette mutagenesis.^[2] Regardless of the source of the DNA library, it is ligated into the appropriate place in the relevant plasmid/phagemid using the appropriate restriction endonucleases.^[2]

E. coli-specific considerations

By placing the hybrid proteins under the control of IPTG-inducible lac promoters, they are expressed only on media supplemented with IPTG. Further, by including different antibiotic resistance genes in each genetic construct, the growth of non-transformed cells is easily prevented through culture on media containing the corresponding antibiotics. This is particularly important for counter selection methods in which a lack of interaction is needed for cell survival.^[2]

The reporter gene may be inserted into the E. coli genome by first inserting it into an episome, a type of plasmid with the ability to incorporate itself into the bacterial cell genome^[2] with a copy number of approximately one per cell.^[7]

The hybrid expression phagemids can be electroporated into E. coli XL-1 Blue cells which after amplification and infection with VCS-M13 helper phage, will yield a stock of library phage. These phage will each contain one single-stranded member of the phagemid library.^[2]

Recovery of protein information

Once the selection has been performed, the primary structure of the proteins which display the appropriate characteristics must be determined. This is achieved by retrieval of the protein-encoding sequences (as originally inserted) from the cells showing the appropriate phenotype.

E. coli

The phagemid used to transform E. coli cells may be "rescued" from the selected cells by infecting them with VCS-M13 helper phage. The resulting phage particles that are produced contain the single-stranded phagemids and are used to infect XL-1 Blue cells.^[2] The double-stranded phagemids are subsequently collected from these XL-1 Blue cells, essentially reversing the process used to produce the original library phage. Finally, the DNA sequences are determined through dideoxy sequencing.^[2]

Controlling sensitivity

The Escherichia coli-derived Tet-R repressor can be used in line with a conventional reporter gene and can be controlled by tetracycline or doxicycline (Tet-R inhibitors). Thus the expression of Tet-R is controlled by the standard two-hybrid system but the Tet-R in turn controls (represses) the expression of a previously mentioned reporter such as HIS3, through its Tet-R promoter. Tetracycline or its derivatives can then be used to regulate the sensitivity of a system utilising Tet-R.^[1]

Sensitivity may also be controlled by varying the dependency of the cells on their reporter genes. For example, this effected by altering the concentration of histidine in the growth medium for his3-dependent cells and altering the concentration of streptomycin for aadA dependent cells.^[3]^[2] Selection-gene-dependancy may also be controlled by applying an inhibitor of the selection gene at a suitable concentration. 3-Amino-1,2,4-triazole (3-AT) for example, is a competitive inhibitor of the HIS3-gene product and may be used to titrate the minimum level of HIS3 expression required for growth on histidine-deficient media.^[2]

Sensitivity may also be modulated by varying the number of operator sequences in the reporter DNA.

Non-fusion proteins

A third, non-fusion protein may be co-expressed with the two fusion proteins. Depending on the investigation, the third protein may modify one of the fusion proteins or mediate or interfere with their interaction.^[1]

Coexpression of the third protein may be necessary for modification or activation of one or both of the fusion proteins. For example S. cerevisiae possesses no endogenous tyrosine kinase. If an investigation involves a protein that requires tyrosine phosphorylation, the kinase must be supplied in the form of a tyrosine kinase gene.^[1]

The non-fusion protein may mediate the interaction by binding both fusion proteins simultaneously, as in the case of ligand-dependent receptor dimerisation.^[1]

For a protein with an interacting partner, its functional homology to other proteins may be assessed by supplying the third protein in non-fusion form which then may or may not compete with the fusion-protein for its binding partner. Binding between the third protein and the other fusion protein will interrupt the formation of the reporter expression activation complex and thus reduce reporter expression, leading to the distinguishing change in phenotype.^[1]

One-hybrid, three-hybrid and one-two-hybrid variants

One-hybrid

The one-hybrid variation of this technique is designed to investigate protein-DNA interactions and uses a single fusion protein in which the AD is linked directly to the binding domain. The binding domain in this case however is not necessarily of fixed sequence as in two-hybrid protein-protein analysis but may be constituted by a library. This library can be selected against the desired target sequence which is inserted in the promoter region of the reporter gene construct. In a positive-selection system, a binding domain which successfully binds the UAS and allows transcription will thus be selected.^[1]

Note that selection of DNA-binding domains isn't necessarily performed using a one-hybrid system, but may also be performed using a two-hybrid system in which the binding domain is varied and the bait and prey proteins are kept constant.^[2]^[3]

Three-hybrid

RNA-protein interactions have been investigated through a three-hybrid variation of the two-hybrid technique. In this case, a hybrid RNA molecule serves to adjoin together the two protein fusion domains which aren't intended to interact with each other but rather the intermidiary RNA molecule (through their RNA-binding domains).^[1] Techniques involving non-fusion proteins that perform a similar function, as described in the 'non-fusion proteins' section above, may also be referred to as three-hybrid methods.

One-two-hybrid

Simultaneous use of the one- and two-hybrid methods (that is, simultaneous protein-protein and protein-DNA interaction) is known as a one-two-hybrid approach and expected to increase the stringency of the screen.^[1]

Host organism

Although theoretically, any living cell might be used as the background to a two-hybrid analysis, there are practical considerations that dictate which will be chosen. The chosen cell line should be relatively cheap and easy to culture and sufficiently robust to withstand application of the investigative methods and reagents.^[1]

[edit] Yeast

S. cerevisiae was the model organism used during the two-hybrid technique's inception. It has several characteristics that make it a robust organism to host the interaction, including the ability to form tertiary protein structures, neutral internal pH, enhanced ability to form disulphide bonds and reduced-state glutathione among other cytosolic buffer factors, to maintain a hospitable internal environment.^[1] The yeast model can be manipulated through non-molecular techniques and its complete gene sequence is known.^[1] Yeast systems are tolerant of diverse culture conditions and harsh chemicals which could not be applied to mammalian tissue cultures.^[1]

Proteins from as small as eight to as large as 750 amino acids have been studied using yeast.^[1]

E. coli

E. coli-based methods have several characteristics which may make them preferable to yeast-based homologues. The higher transformation efficiency and faster rate of growth lends E. coli to the use of larger libraries (in excess of 10⁸).^[2] A low false positive rate of approximately 3x10⁸, the absence of requirement for a nuclear localisation signal to be included in the protein sequence and the ability to study proteins that would be toxic to yeast may also be major factors to consider when choosing an experimental background organism.^[2]

It may be of note that the methylation activity of certain E. coli DNA methyltransferase proteins may interfere with some DNA-binding protein selections. If this is anticipated, the use of an E. coli strain that is defective for a particular methyltransferase may be an obvious solution.^[2]

Applications

Determination of sequences crucial to interaction

By changing specific amino acids by mutating the corresponding DNA base-pairs in the plasmids used, the importance of those amino acid residues in maintaining the interaction, can be determined.^[1]

After using bacterial cell-based method to select DNA-binding proteins, it is necessary to check the specificity of these domains as there is a limit to the extent to which the bacterial cell genome can act as a sink for domains with an affinity for other sequences (or indeed, a general affinity for DNA).^[2]

Drug and poison discovery

Protein-protein signalling interactions pose suitable therapeutic targets due to their specificity and pervasiveness. The random drug discovery approach utilises compound banks that comprise random chemical structures and demands a high-throughput method in order to test these structures in their intended target.^[1]

The cell chosen for the investigation can be specifically engineered to mirror the molecular aspect that the investigator intends to study and then used to identify new human or animal therapeutics or anti-pest agents.^[1]

Determination of protein function

By determination of the interaction partners of unknown proteins, the possible functions of these new proteins may be inferred.^[1] This can be done using a single known protein against a library of unknown proteins or conversely, by selecting from a library of known proteins using a single protein of unknown function.^[1]

Zinc finger protein selection

To select zinc finger proteins (ZFPs) for protein engineering, methods adapted from the two-hybrid screening technique have been used with success.^[3]^[2] A ZFP is itself a DNA-binding protein used in the construction of custom DNA-binding domains that bind to a desired DNA sequence.^[8]

By using a selection gene with the desired target sequence included in the UAS and randomising the relevant amino acid sequences to produce a ZFP library, cells that host a DNA-ZFP interaction with the required characteristics can be selected. Each ZFP typically recognises only 3-4 base pairs, so to prevent recognition of sites outside the UAS, the randomised ZFP is engineered into a 'scaffold' consisting of another two ZFPs of constant sequence. The UAS is thus designed to include the target sequence of the constant scaffold in addition to the sequence for which a ZFP is being selected.^[3]^[2]

A number of other DNA-binding domains may also be investigated using this system.^[2]

Strengths and weaknesses

Two-hybrid screens are now routinely performed in many labs. They can provide an important first hint for the identification of interaction partners. Moreover, the assay is scalable which makes it possible to screen for interactions among many proteins.

The main criticism applied to the yeast two-hybrid screen of protein-protein interactions is the possibility of a high number of false positive (and false negative) identifications. The exact rate of false positive results is not known, but estimates are as high as 50%.^[9] The reason for this high error rate lies in the principle of the screen: The assay investigates the interaction between (i) overexpressed (ii) fusion proteins in the (iii) yeast (iv) nucleus. Each of these points (i-iv) alone can give rise to false results. For example, overexpression can result in non-specific interactions. Moreover, a mammalian protein is sometimes not correctly modified in yeast (e.g. missing phosphorylation), which can also lead to false results. Finally, some proteins might specifically interact when they are co-expressed in the yeast, although in reality they are never present in the same cell at the same time. Due to the combined effects of all error sources the overall confidence of the yeast two-hybrid assay is rather low. This means that all interactions have to be confirmed by a high confidence assay like co-immunoprecipitation of the endogenous proteins. However, this is a difficult task, especially for large scale protein-protein interaction data.

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