My Life

Kitne Sooraj ko dubte dekha hai
Chand ko roshni dete dekha hai
Par kabhi ye nai socha tha
Ki har muskil ka koi rasta nikalta hai

Bachpan ki wo choti choti baton pe ladayi kiya hai
Gharwalon se mar bhi khaya hai
Par kabhi ye nai socha tha
Ki har galti se kuch sikha hai

Jab kabhi dard hota tha
to wo pyaar apnon ka milta tha
Par kabhi ye nai socha tha
Ki Zindagi ke dard ko khud hi samjhna hai

Man me koi chah ho to hoton tak aa jata tha
Aur wo khwaish apne pura karte the
par kabi ye nai socha tha
Ki zindagi ke aste me bahut si betabiyaan rehti hai

Story of 3 Idiots

This story started when I entered into one of the premier institutes of India named Vellore Institute of Technology (VIT). I hope all my readers know this name as I don’t want to elaborate on this. The entrance into this itself is a story.
1. My Entrance – I was completely confused and shattered after I finished my graduation as I was holding no jobs in my hand as well as I didn’t had enough money to think about studying abroad. So everyday my mom used to pray for me that somewhere someday I will get a call for a good opportunity. After completing my graduation I left for my home at Kolkata along with a group of four friends. One of my friend came to station to sea off me. He filled the application form for VIT entrance exam and asked me too to fill the form. So I asked him to pay the money for me and apply for the exam which he did. After coming home daily I started preparing for NET exam which is the toughest exam to crack. The exam went very bad as I was unable to answer any question in Part-1 itself. All droppers and professors appeared in that exam. I was looking like a big fool in front of them. After that exam went away my hopes for my future became more tensed as I was having nothing in my head. One fine day, my mobile beeped and I got a message. It said that “ Your VIT entrance exam is tomorrow at 10 AM at this venue and you will be provided admit card half an hour before the exam. So please come an hour before the exam”. I was completely in a haphazard situation. My mom told this may be your last chance. Just give a try and write the exam. Next day, I went for this exam. The traffic at Kolkata made me enter into the institute 5 minutes late into the examination hall. I just took my admit card in a hurry and entered into the hall with complete sweat. The paper was quite easy and I finished my paper half an hour before the bell ranged and was enjoying butterscotch ice-cream outside the institute. One fine day I got a message which said that I qualified the exam and was asked to come to VIT for counseling.
2. Selection Day - On the counseling day I was totally haphazard after losing my seat in biotech. I was confused between nanotech and environmental engineering. One of my friends from my graduation also came for the counseling and she advised me to take environmental engineering which I did. After I finished my counseling, I was thinking that I have done a big mistake. I left for my home by taking a flight next day.
3. First Day at VIT: As it was my first day, I was very tensed about the kind of class, teachers and friends with whom I have to stay here for 2 years. As I entered into the class, I saw all dull and ugly faces except 2-3 and all my hopes were shattered. I sat at third row second bench along with some unknown boys. After one period of class, I started talking with some of them but I couldn’t find that compatibility which makes me to talk again. The day finished and I was hoping that with passage of time I may get some good friends.
4. First Idiot: It was evening time I was feeling bored so I just thought of going to library to issue some good books. I went to library and there I met my FIRST IDIOT who was sitting on a chair and was reading some biotech books sincerely. As he was my classmate I approached to him and just had a formal talk with him. I told him about my whereabouts and my interests and we started talking for a long time. He encouraged me to take up some good biotech topics which we can apply in our field and carry out some good projects here. We exchanged numbers and asked each other’s room number. So that day I felt like that I met a friend who is funny, kiddish, jovial, knowledgeable and full of masti type. He was from Mumbai, especially MULUND which he thinks the best place in the world.
5. Second Idiot: As the days were passing, I started talking with almost all the boys. Out of which there was one bald-headed, low vest pant wala whose red underwear always was the highlight just started talking with me. He told me that he already has done 2 jobs and he was the first person to take this branch. He was from Kalyan, Mumbai. We didn’t shared such close rapport as he use to be in his own world but was very friendly with anybody. He gave me his number and we started sitting with each other in the class.
6. Third Idiot: One fine day, a new girl from Jammu who was so fair entered into the class as she joined late and quietly sat at the back bench. I asked my first idiot who is she. He jokingly said that I already know her. She is from Jammu. Then I got a message from my graduation friend asking “ What happened? Wo nayi ladki ko dekh ke hosh ud gaye kya??” I replied “ Waise dikhne me to mast hai…..par lagta hai sharmili type ki hai…pata nai bat karegi ki nahi??”. Then in second half we had lab classes where I saw her talking to a group of friends. I interfered in their talks and asked her something for which she replied in a very funny way. As I was listening to her all my imaginations about her went to haste. Because she was totally opposite to what I was thinking about her. One fine day she messaged me about some help and in this way I got her number and started messaging and talking in the class.
7. First Semester: The first semester we enjoyed a lot. We had lot of class parties and outings. The whole class was divided into groups and they use to enjoy in groups. My group comprised of tehse 3 idiots along with some other friends. During exam time, me along with these 2 idiots use to study together and the third idiot use to be over phone since we had separate boys and girls hostels ( God…..Y can’t we have common hostels???).. So, in this way we use to clear each other’s doubt and use to help each other. When we boys failed to understand anything, we use to call that third idiot in library and use to study together. ( Frankly speaking….We had almost 80% masti and 20 % study…..because that third idiot use to laugh like anything….the faces she makes and clueless smiles makes everybody to join her). We had a complete masti all the time.
8. Second Semester: As we were entering into the second phase, our subjects increased and pressure increased. But then who cares…at last we have to study for one night before the exam. The whole class went for Nagpur trip which we enjoyed a lot. We presented our posters at an international conference in which we had parties, DJ Night and so much fun. After coming from the trip we were busy finishing our assignments and exam preparations. We studied like anything to clear the exams.
9. Third Semester: The third semester was having less subjects and so were quite relaxed. In between we had some quarrel between the 2 idiots as we all planned to stay in a single room which I planned but later on I dumped them because of some confusion and fight with my roommates. I really feel bad for it still now as it made them helpless and till now we discuss on that thing. But later on everything was settled. We all were in the same hostel. And this new game invented by my MULUND friend which was “MAFIA” and it was so interesting that at late nights we use to assemble in someone’s room and kept playing for late nights. The god in that game was constant which the first idiot I met was. I used to be easily caught in that game as I am very bad in acting. But it was fun and we use to laugh a lot.
10. Seperation: The fourth semester was a separation semester. We were supposed to do projects for a period of 7-8 months and everybody were applying outside college as it will be useful in future. One of my idiot friends from Kalyan already got the project at AMPRI Bhopal through my HOD’s recommendation. So, he was decided that he will leave for Bhopal. But me along with my two idiots were still passing through a tensed situation about our project work. Then one fine day, my dad told me about a company named Ramky and I told to my friend from mulund about this. I didn’t had any hopes from this company as I took it lightly. At class, my jammu friend asked me and I told her about this company. My father gave me the HR’s mail id and number to which we 3 of us sent our resumes and applied for it. It was under process for almost one month and we were on continuous conversation with the HR. One fine day we were supposed to give telephonic interview for which call came for me along with my Jammu friend. My third friend didn’t got chance for which we felt very bad as we all applied together. But that’s life…we accepted and my friend who likes Mumbai very much said that my luck is always in Mumbai and I will definitely get there. The happiest moment came when I was studying for my last semester paper and my Jammu friend called me up and told me that we will be getting stipend during the course of our internship. I was so excited. The paper also went well and I was very happy.
11. Journey To Hyderabad: Before leaving for our internship one company named Cognizant came to our campus for placement which we applied. In the aptitude test my 2 best buddies didn’t got chance to face interview. My interview went very bad and I was having no hopes at all but finally me along with that Jammu friend got selected in the company. The very next day along with her I left for Hyderabad. We were excited that what kind of company will be….how we will stay there….etc etc….but being with her all my worries are nothing…because whenever I am with her I am so lucky that I surpass all my difficulties. She is a good luck charm for me. In the train we had long discussions about our journey so far in life and were planning about our stay in Hyderabad.
12. Entrance to Hyderabad: As we reached Hyderabad, the company gave us guest houses for a temporary stay. We went straight to the guest house. As we entered the guest house we were enthralled with the ambience and the maintenance of the guest house. The rooms were well furnished and all the facilities were there except food. After fresh up we went to company and were equally excited.
13. First Day at Office: My first day in office was very exciting. We were warmly welcomed by the company members. We met Vice-President of the company and he was very jovial in his interaction with us. We never felt like we are treated as students here. We were given separate cubicles along with net connection. Our project guide was very helpful in deciding our topic of project and we finally decided our topics after some days. The moment I logged in my net connection I checked facebook page which was blocked. The next thing I did I created a Gmail acc for her and dragged her into chats. We use to have long chats and whenever we need something we use to ask in chats. Its later on we discovered that there are extension phones on which we can speak. That day I dragged her to have our lunch at KFC which was nearby our office and we enjoyed our meal.
14. Life at Hyderabad: We came to Hyderabad at the month of September and for 2 weeks we were at guest houses. But the real struggle which we faced was getting a paying guest to stay at Hyderabad because after 2 weeks we need to leave the guest house. So we started roaming like helpless fellows, went here and there and scanned entire Hyderabad for getting a good guest house but we failed to get a single good room. After days of our struggle the caretaker of guest house had some broker friend who showed me a paying guest which was good and I took the room. But still I was trying for her to get a good paying guest. We talked to a lot of brokers and finally we managed to get a good paying guest for her. So now our lives were on track.
15. Problems We faced: The problem which I faced was about food and I use to daily complain about the food I use to get for my lunch. But I didn’t had any choice so I have to stay there. Daily I use to tell her that what problems I am facing and she use to laugh at me and use to say that everything will be fine. The problem which she faced was she use to get cockroaches in her food…hahahaaaa……and she left that paying guest and shifted into a flat with a Bengali roommate. ( The bong connection will always be with her) So now on she use to cook food and use to eat and I use to get my BAKWAS food from my PG and use to share her food. One day she brought “Anda Curry” for me. I asked for 2 eggs. It was delicious. After having lunch we were full and planned to take half day’s leave and we packed everything ..took auto and just escaped.
16. Most Important Day: One fine day at office she asked me to come to her and she showed me the mail which our college sent. The mail said that Ramky is coming for campus recruitment and we were very much excited. We told our friends and the other 2 idiots too. Out of that my mulund friend was not eligible for this….too bad. So we were asking here and there that which division is coming and weather its Hyderabad division. Later on we explored that its from the other division of Ramky which is opposite to our division at Hyderabad itself. We went to the HR and he was very excited to know that we are from VIT and doing intern here. So, he told us about the entire process and he asked us to go through the normal process for which we have to go to VIT and finish the procedure. We went to college, finished the aptitude test and interview and came back to Hyderabad. After almost 2 weeks we got the results in which we were selected but our 2nd idiot from Kalyan missed the chance which was again a setback for us. We felt very bad for him as if he would have been here we would have regained our group again. So now its me and my jammu friend who has been a common factor for me always and my lucky charm.

Our joining is on June and after one month training we will be given postings. Donno what is written in the future but I don’t want to miss this third idiot in my life.

THE JOURNEY CONTINUES……….

DEVELOPMENT OF TISSUE ENGINEERED XENOGRAFTS

Heart valve diseases have a significant high mortality, and the valve replacement using glutaraldehyde crosslinked xenografts is one of the main curing techniques. But its application is limited due to poor durability, calcification of the valves and immunogenic reactions. Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes [galactose-a-(1,3)-galactose terminal disaccharide (a-Gal)]. Tissue-engineered or decellularized xenografts and homografts have already been implanted in humans or are currently approaching the clinical setting. However, the success of tissue-engineering procedures depends on the choice of an appropriate biomaterial. Decellularized xenogenic tissue potentially combines the structural composition of the targeted tissue with a reduced risk of graft rejection or disease transmission. In this study, we will test the effectiveness of currently available decellularization methods based on TRITON-X, sodium dodecyl sulfate (SDS), and trypsin. After identifying the most effective decellularization method, the tissue will undergo different novel crosslinking reagents and subsequently the capacity for reseeding with endothelial cells will be studied.

Nanorobots in anasthesiology

NANOTECH CONTROLLER
Nanotechnology will let us build fleets of computer controlled molecular tools and by
using this nano materials are created which these materials have very different properties than
materials at nano scale. They can be stronger, lighter, more electrically conductive, more
porus and less corressive than bulk materials. The nanomaterials have the potential to solve
unique biological challenges not currently possible.
The common examples of nanomaterials are found in scientific literature flurescence,
nanotubes, buckballs, quantum dots and nanoshells.
This was coined in1974 by Norio Taniguchi at the University of Tokyo.
Nanotechnology is actually a multitude of rapidly emerging technologies, most promising
potential of nanotechnology exists due to the laws of quantum physics.
Quantum physics laws take over at this scale enabling novel applications in optics,
electronics, magnetic storage, computing, catalysts and other areas.
It uses a basic unit of measure called a“nanometer” ( abbreviated nm) derived from
greek word for midget ,”nano” is a metric prefix and indicates a billionth part (10-9).There
are one million nm’s to ammeter
Each nm is only three to five atoms wide. They’re small. Really ~40,000 times smaller than
the width of an average human hair.
The invention of super small computers bacteria sized with todays MIPS capacity
processing power of billion laptops.
A nanometer is one thousandth of a micron and a thousandth of a millionth of meter
(a billionth of a meter). Imagine one billion nanometer in a meter perspective a nanometer. Is
about the width of six bounded carbon atoms, and approximately 40,000 are needed to equal
the width of an average human hair.
Another way to initialize nanometer 1 inch =25,400,000 nanometres.
A red blood cell is ~ 7,000 nm in diameter. And ~2,000 nm in height.
A virus is ~ 100 nm.
A buck ball is 1 nm.
A hydrogen atom is 1 nm.
For our purposes nanometers pertains to science, technology, manufacturing,
chemistry, health science, space programs, medical and engineering.
In engineering , it is used to reduce the size and increase the efficiency . But , in
medical it is used to overcome operations and severe risk taking disease. Think of your brain,
Now performing vastly superior levels Nano dots will become an as-needed addition to your
existing neurons extending your mental capabilities further than imagine .
The tablets, injections are used in nano technology for curing disease for living
organisms.
“But,in this tablets and injections are used for giving
instructions in brain ,and also we can be safe from any disease and we can cure any heart or
kidney problems without any surgery . During this treatment the human can continue his/her
regular work as usual. This can be taken as tablet, injection, food, cool drinks, oil applied to
hair etc”. for this construction a computer and the nano medicine is used.
Introduction
Background
The medical area of nano science application is one of the most potentially valuable, with
many projected benefits to humanity. Cells themselves are very complex and efficient nanomachines,
and chemists and biochemists have been working at the nano scale for some time
without using the nano label. Some areas of nano science aim to learn from biological nano
systems, while others are focusing on the integration of the organic and inorganic at the nano
scale. Many possible applications arising from this science are being researched.
Drug Delivery Using Nanoparticles and Molecular Carriers
Finally, drug delivery is likely to benefit from the development of nanotechnology.
With nanoparticles it is possible that drugs may be given better solubility, leading to better
absorption. Also, drugs may be contained within a molecular carrier, either to protect them
from stomach acids or to control the release of the drug to a specific targeted area, reducing
the likelihood of side effects. Such drugs are already beginning pre-clinical or clinical trials,
adhering to the strict regulatory requirements for new pharmaceuticals. Due to this,
development costs are often high and outcomes of research sometimes limited.
Lab on a Chip and Advanced Drug Delivery Systems
The ultimate combination of the laboratory-on-a-chip and advanced drug delivery
technologies would be a device that was implantable in the body, which would continuously
monitor the level of various biochemicals in the bloodstream and in response would release
appropriate drugs. For example, an insulin-dependent diabetic could use such a device to
continuously monitor and adjust insulin levels autonomously. There is no doubt that this is
the direction that current advances in which micro fluidics and drug delivery are heading.
Anesthesia
Anesthesia, or anaesthesia has traditionally meant the condition of having sensation
(including the feeling of pain) blocked. This allows patients to undergo surgery and other
procedures without the distress and pain they would otherwise experience. The word was
coined by Oliver Wendell Holmes, Sr. in 1846. Another definition is a "reversible lack of
awareness", whether this is a total lack of awareness (e.g. a general anaesthestic) or a lack of
awareness of a part of a the body such as a spinal anaesthetic or another nerve block would
cause. Anesthesia differs from analgesia in blocking all sensation, not only pain.
Today, the term general anesthesia in its most general form can include:
• Analgesia: blocking the conscious sensation of pain;
• Hypnosis: produces unconsciousness without analgesia;
• Amnesia: preventing memory formation;
• Relaxation: preventing unwanted movement or muscle tone;
• Obtundation of reflexes, preventing exaggerated autonomic reflexes.
Patients undergoing surgery usually undergo preoperative evaluation. It includes gathering
history of previous anesthetics, and any other medical problems, physical examination,
ordering required blood work and consultations prior to surgery.
There are several forms of anesthesia. The following forms refer to states achieved by
anesthetics working on the brain:
• General anesthesia: "Drug-induced loss of consciousness during which patients are
not arousable, even by painful stimulation." Patients undergoing general anesthesia
can often neither maintain their own airway nor breathe on their own. While usually
administered with inhalational agents, general anesthesia can be achieved with
intravenous agents, such as propofol.
• Deep sedation/analgesia: "Drug-induced depression of consciousness during which
patients cannot be easily aroused but respond purposefully following repeated or
painful stimulation." Patients may sometimes be unable to maintain their airway and
breathe on their own.
• Moderate sedation/analgesia or conscious sedation: "Drug-induced depression of
consciousness during which patients respond purposefully to verbal commands, either
alone or accompanied by light tactile stimulation." In this state, patients can breathe
on their own and need no help maintaining an airway.
• Minimal sedation or anxiolysis: "Drug-induced state during which patients respond
normally to verbal commands." Though concentration, memory, and coordination
may be impaired, patients need no help breathing or maintaining an airway.
The level of anesthesia achieved ranges on a continuum of depth of consciousness from
minimal sedation to general anesthesia. The depth of consciousness of a patient may change
from one minute to the next.
The following refer to the states achieved by anesthetics working outside of the brain:
• Regional anesthesia: Loss of pain sensation, with varying degrees of muscle
relaxation, in certain regions of the body. Administered with local anesthesia to
peripheral nerve bundles, such as the brachial plexus in the neck. Examples include
the interscalene block for shoulder surgery, axillary block for wrist surgery, and
femoral nerve block for leg surgery. While traditionally administered as a single
injection, newer techniques involve placement of indwelling catheters for continuous
or intermittent administration of local anesthetics.
o Spinal anesthesia: also known as subarachnoid block. Refers to a Regional
block resulting from a small volume of local anesthetics being injected into the
spinal canal. The spinal canal is covered by the dura mater, through which the
spinal needle enters. The spinal canal contains cerebrospinal fluid and the
spinal cord. The sub arachnoid block is usually injected between the 4th and
5th lumbar vertebrae, because the spinal cord usually stops at the 1st lumbar
vertebra, while the canal continues to the sacral vertebrae. It results in a loss of
pain sensation and muscle strength, usually up to the level of the chest (nipple
line or 4th thoracic dermatome).
o Epidural anesthesia: Regional block resulting from an injection of a large
volume of local anesthetic into the epidural space. The epidural space is a
potential space that lies underneath the ligamenta flava, and outside the dura
mater (outside layer of the spinal canal). This is basically an injection around
the spinal canal.
• Local anesthesia is similar to regional anesthesia, but exerts its effect on a smaller
area of the body.
History
Herbal derivatives
The first anesthesia (a herbal remedy) was administered in prehistory. Opium poppy capsules
were collected in 4200 BC, and opium poppies were farmed in Sumeria and succeeding
empires. The use of opium-like preparations in anaesthesia is recorded in the Ebers Papyrus
of 1500 BC
Non-pharmacological methods
Hypnotism and acupuncture have a long history of use as anesthetic techniques. In China,
Taoist medical practitioners developed anesthesia by means of acupuncture. Chilling tissue
(e.g. with ice) can temporarily cause nerve fibers (axons) to stop conducting sensation, while
hyperventilation can cause brief alteration in conscious perception of stimuli including pain
(see Lamaze).
Anesthetic agents
Local anesthetics
inhaled general anesthetic agents
intravenous anesthetic agents (non-opioid)
Current intravenous opioid analgesic agents
Current muscle relaxants
intravenous reversal agents
Anesthetic equipment
In modern anesthesia, a wide variety of medical equipment is desirable depending on the
necessity for portable field use, surgical operations or intensive care support. Anesthesia
practitioners must possess a comprehensive and intricate knowledge of the production and
use of various medical gases, anaesthetic agents and vapours, medical breathing circuits
and the variety of anaesthetic machines (including vaporizers, ventilators and pressure
gauges) and their corresponding safety features, hazards and limitations of each piece of
equipment, for the safe, clinical competence and practical application for day to day practice.
Anesthetic monitoring
Patients being treated under general anesthetics must be monitored continuously to ensure the
patient's safety.
Anesthesia record
The anesthesia record is the medical and legal documentation of events during an anesthetic.
It reflects a detailed and continuous account of drugs,
NANOTECHNOLOGY::
Nano is one billionth of one. Now we have the so-called microprocessors
and microarray technology that would reach the nano level within a few decades, we suppose.
Some call this technology to be nanotechnology and some others name it the molecular
nanotechnology, to be specific.
REASONS FOR APPLYIING NANOTECH TO BIIOLOGIICAL SYSTEMS::
Most animal cells are 10,000 to 20,000 nanometers in diameter. This means that
nanoscale devices (having at least one dimension less than 100 nanometers) can enter cells
and the organelles inside them to interact with DNA and proteins. Tools developed through
nanotechnology may be able to detect disease in a very small amount of cells or tissue. They
may also be able to enter and monitor cells within a living body. Miniaturization will allow
the tools for many different tests to be situated together on the same small device. This
means that nanotechnology could make it possible to run many diagnostic tests
simultaneously as well as with more sensitivity. In general, nanotechnology may offer a
faster and more efficient means for us to do much of what we do now.
NANOMEDICINE::
The emerging field of nanorobotics is aimed at overcoming the
shortcomings present in the traditional way of treatment of patients. Our bodies are filled
with intricate, active molecular structures. When those structures are damaged, health
suffers. Modern medicine can affect the work of the body in many ways, but from a
molecular viewpoint it remains crude. Molecular manufacturing can construct a range of
medical instruments and devices with greater abilities. The human body can be seen as a
workyard, construction site, and battleground form molecular machines. It works
remarkably well; using systems so complex that medical science still doesn’t understand
many of them.
BIOMEDICAL APPILICATIONS OF NANOROBOTS::
The enormous potential in the biomedical capabilities of nanorobots and
the imprecision and side effects of medical treatments today make nanorobots very
desirable. But today, in this revolutionary era we propose for nanomedical robots, since
they will have no difficulty in identifying the target site cells even at the very early stages
which cannot be done in the traditional treatment and will ultimately be able to track them
down and destroy them wherever they may be growing. By having these Robots, we can
refine the treatment of diseases by using biomedical, nanotechnological engineering.
Nanorobot designed to perform cell surgery
WHAT IS A MEDICINAL NANOROBOT ?
Nanorobots are theoretical microscopic devices measured on the scale of nanometers (1
nm equals one millionth of a millimeter). When fully realized from the hypothetical stage, they
would work at the atomic, molecular and cellular level to perform tasks in both the medical and
industrial fields that have heretofore been the stuff of science fiction Nanomedicine’s nanorobots
are so tiny that they can easily traverse the human body. Scientists report the exterior of a
nanorobot will likely be constructed of carbon atoms in a diamondoid structure because of its
inert properties and strength. Super-smooth surfaces will lessen the likelihood of triggering the
body’s immune system, allowing the nanorobots to go about their business unimpeded. Glucose
or natural body sugars and oxygen might be a source for propulsion, and the nanorobot will have
other biochemical or molecular parts depending on its task.
Nanorobot in Nanoscale
According to current theories, nanorobots will possess at least rudimentary two-way
communication; will respond to acoustic signals; and will be able to receive power or even reprogramming
instructions from an external source via sound waves. A network of special
stationary nanorobots might be strategically positioned throughout the body, logging each active
nanorobot as it passes, then reporting those results, allowing an interface to keep track of all of
the devices in the body. A doctor could not only monitor a patient’s progress but change the
instructions of the nanorobots in vivo to progress to another stage of healing. When the task is
completed, the nanorobots would be flushed from the body.
Nanorobot performing operations on blood cells
IMPLEMENTATION::
Anestthesiia USING NANOTECHNOLOGY::
Automated anesthesia delivery.
Automated delivery of inhalational anesthetics.
Automated I.V. anesthesia delivery system.
Nano assisted titration of I.V. agents with target controlled infusion.
CREATIION OF NANO DEVIICES::
The creation of the nano devices can be done using any of the two techniques that
are available. They are
• Top-down approach
• Bottom-up approach
CHALLENGES FACED BY NANOROBOTS::
While designing nonorobots in nanoscale dimensions there should be a better
understanding of how matter behaves on this small scale. Matter behaves differently on the
nanoscale than it does at larger levels. So the behaviour of the nanorobots must be taken
care so that the do not affect us both inside and outside the body.
Other challenges apply specifically to the use of nanostructures within biological
systems. Nanostructures can be so small that the body may clear them too rapidly for them
to be effective in detection or imaging. Larger nanoparticles may accumulate in vital
organs, creating a toxicity problem. So we need to consider these factors as they anticipate
how nanostructures will behave in the human body and attempt to create devices the body
will accept.
DESIIGN OF NANOROBOTS::
The nanorobots that we describe here will be floating freely inside the body
exploring and detect the various receptors eg GABA receptors in the brain, opioid
receptors, neuromuscular junction receptors. So, while designing such a nanorobot for
anesthesia, the main factors that are to be considered are given below.
TECHNIIQUE USED::
We use the bottom-up approach, which involves assembling structures atom-byatom
or molecule-by-molecule which will be useful in manufacturing devices used in
medicine.
SIIZE::
Nanorobots will typically be .5 to 3 microns large with 1-100 nm parts. Three microns
is the upper limit of any nanorobot because nanorobots of larger size will block capillary
flow.
STRUCTURE::
The nanorobot’s structure will have two spaces that are
Interior:
It will be a closed, vacuum environment into which liquids from the outside cannot
normally enter unless it is needed for chemical analysis.
Exterior: It will be subjected to various chemical liquids in our bodies.
CHEMIICAL ELEMENTS::
Carbon will likely be the principal element comprising the bulk of a
medical nanorobot, probably in the form of diamond or diamondoid/fullerene
nanocomposites largely because of the tremendous strength and chemicalinertness of
diamond. Many other light elements such as hydrogen, sulfur, oxygen,nitrogen, fluorine,
silicon, etc. may also be used
ABIILIITY TO DEFEND FROM IIMMUNE SYSTEM::
Immune system response is primarily a reaction to a "foreign" surface..
Passive diamond exteriors may turn out to be ideal. Several experimental studies hint that
the smoother and more flawless the diamond surface, the less leukocyte activity and the less
fibrinogen adsorption we will get. So it seems reasonable to hope that when diamond
coatings can be laid down with almost flawless atomic precision, making nanorobot exterior
surfaces with near-nanometer smoothness that these surfaces may have very low
bioactivity. Due to the extremely high surface energy of the passivated diamond surface and
the strong hydrophobicity of the diamond surface, the diamond exterior is almost
completely chemically inert and so opsonization should be minimized. If flawless diamond
surfaces alone do not prove fully bioinactive as hoped, active surface management of the
nanorobot exterior can be used to ensure complete nanodevice biocompatibility. Allergic
and shock reactions are similarly easily avoided.
ACQUIIRING POWER::
It could metabolize local glucose and oxygen for energy. Another possibility is
externally supplied acoustic power, which is probably most appropriate in a clinical setting.
There are literally dozens of useful power sources that are potentially available in the
human body.
COMMUNIICATON::
Having nanorobots inside the body it is very essential to know the actions done by it.
There are many different ways to do this. One of the simplest ways to send broadcast-type
messages into the body, to be received by nanorobots, is acoustic messaging. A device
similar to an ultrasound probe would encode messages on acoustic carrier waves at
frequencies between 1-10 MHz.
TRACKIING::
A navigational network may be installed in the body, with stationkeeping
navigational elements providing high positional accuracy to all passing nanorobots
that interrogate them, wanting to know their location. Physical positions can be
reported continuously using an in vivo communications network.
STRUCTURE OF NANOROBOT::
The nanorobot consists of three main parts like the receptor sensor, CPU, effector and the
power system. The purpose of receptor sensor is to identify the different anesthesia receptors on
the cell. The effector is used to produce the post receptor event.The CPU controls all the activities
.The power system provides the necessary energy for the working of the nanorobot.
GP120
layer GABA
layer
REQIREMENTS OF THE NANOROBOT:
1. It should e very small so that the blood capillary flow is not affected.
2. It should not be affected by the WBC.
3. It should be capable of attaching to anesthesia receptors only.
4. It should make its operations in the brain with GABA receptors, in the muscles with
neuromuscular junction and in the spinal cord with the opioid receptors .
5. It should be made of cheaper rates, so that the patient can afford it easily.
OPERATION::
The designed anesthesia nanorobots are injected into the blood stream. These nanorobots attaches
to the various receptor in the different parts of the body and produces the effects.
GABA receptors produces the loss of consciousness and amesia
Neuromuscular junction produces the full muscle relaxation
These two gives the good intubation conditions for securing the airway.
Opioid receptors produce the good analgesia.
In Spinal cord attaches to the sodium channel receptor and produces the spinal anesthesia
Arrangement
to produce
the effect
Recept
or
Power System
Central
Processin
g Unit
effector
Site for
receptor
attachment
ADVANTAGES::
1. More than million people in this world are undergoing surgery where anesthesia is essential.
Currently an anesthesiologist is required to give the anesthesia and carefull titration of the
drugs is essential to prevent the side effects like hypotension, desaturation, preventing the
intubation response.
2. As the nanorobot do not generate any harmful activities there is no side effect. It operates at
specific site only.
3. The initial cost of development is only high but the manufacturing by batch processing
reduces the cost.
4. Can be used in both general as well as spinal anesthesia.
5. Reduces the mortality and morbidity associated with anesthesia.
6. Patient satisfaction.
7. Less drug consumption and hence less side effects.
8. No peaks and downs in pain relief.
9. Labour analgesia; can provide pain relief for the mother at the time of delivery.
DISADVANTAGES::
1.The nanorobot should be very accurate, otherwise harmful effects may occur.
2.The initial design cost is very high.
3.The design of this nanorobot is a very complicated one
CONCLUSION::
The paper is just a recent advancement in the field of nanotechnology gives the hope of
the effective use of this technology in medical field. This paper gives an idea of giving exhalent
pain relief to millions of patients who undergo various types of surgery and also pain relief to
terminally ill cancer patients. Using this technology we can conduct safe and painless delivery
and provide exhalent care to the mother and baby.

Use of DNA barcodes to identify flowering plants

Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.

angiosperm internal transcribed spacer Plummers Island species identification trnH-psbA
The identification of animal biological diversity by using molecular markers has recently been proposed and demonstrated on a large scale through the use of a short DNA sequence in the cytochrome c oxidase 1 (CO1) gene (1-5). These “DNA barcodes” show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, and forensic analysis. Engineered DNA sequences also have been suggested as exact identifiers and intellectual property tags for transgenic organisms (6). A Consortium for the Barcode of Life (http://www.barcoding.si.edu/) has been established to stimulate the creation of a database of documented and vouchered reference sequences to serve as a universal library to which comparisons of unidentified taxa can be made. Here, we propose two DNA regions for barcoding plants and provide an initial test of their utility.

DNA barcoding follows the same principle as does the basic taxonomic practice of associating a name with a specific reference collection in conjunction with a functional understanding of species concepts (i.e., interpreting discontinuities in interspecific variation). Presently, some controversy exists over the value of DNA barcoding (7), largely because of the perception that this new identification method would diminish rather than enhance traditional morphology-based taxonomy, that species determinations based solely on the amount of genetic divergence could result in incorrect species recognition, and that DNA barcoding is a means to reconstruct phylogenies when it is actually a tool to be used largely for identification purposes (8-10). In support of barcoding as a species identification process, Besansky et al. (11), Janzen (12, 13), Hebert et al. (1-4), and Kress (14) have offered arguments for the utility of DNA barcoding as a powerful framework for identifying specimens. Our objective in this paper is not to debate the validity of using barcodes for plant identification, but rather to determine appropriate DNA regions for use in flowering plants.

A portion of the mitochondrial CO1 gene was deliberately chosen for use in animal identification when DNA barcoding was proposed (1), and its broad utility in animal systems has been demonstrated in subsequent pilot studies (1-5). The taxonomic limits of CO1 barcoding in animals are not fully known, but it has proven useful to discriminate among species in most groups tested (2). The choice of a DNA region usable for barcoding has been little investigated in other eukaryotes, whereas in prokaryotes, rRNA genes are favored for identifications (e.g., ref. 15). Among plants, especially angiosperms, DNA-based identifications, although not strictly through the use of DNA barcodes, have been creatively used to reconstruct extinct herbivore diets (16, 17), to identify species of wood (18), to correlate roots growing in Texas caves with the surface flora (19), and to determine species used in herbal supplements (20). However, some of these identifications have not been entirely successful at the species level, and DNA barcoding per se has not yet been applied to plants. The primary reason that barcoding has not been applied to plants by the emerging initiative is that plant mitochondrial genes, because of their low rate of sequence change, are poor candidates for species-level discrimination. The divergence of CO1 coding regions among families of flowering plants has been documented to be only a few base pairs across 1.4 kb of sequence (21, 22). Furthermore, plants rapidly change their mitochondrial genome structure (23), thereby precluding the existence of universal intergenic spacers that otherwise would be appropriately variable unique identifiers at the species level (e.g., ref. 24).

For plant molecular systematic investigations at the species level, the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron (18S-5.8S-26S) is the most commonly sequenced locus (25). This region has shown broad utility across photosynthetic eukaryotes (with the exception of ferns) and fungi and has been suggested as a possible plant barcode locus (26). Species-level discrimination and technical ease have been validated in most phylogenetic studies that employ ITS, and a large body of sequence data already exists for this region (>36,000 angiosperm sequences were available in GenBank in December 2004, although these sequences have not been filtered for taxa, so it is not certain how many species are represented). However, the limitations of this nuclear region in some taxa are well established. ITS has reduced species-level variability in certain groups (especially recently diverged taxa on islands), divergent paralogues that require cloning of multiple copies, and secondary structure problems resulting in poor-quality sequence data (25, 27). In some cases, the preferential amplification of endophytic or contaminating fungi may occur, although this can be eliminated with plant-specific primer design (28, 29).

An advantage of the ITS region is that it can be amplified in two smaller fragments (ITS1 and ITS2) adjoining the 5.8S locus, which has proven especially useful for degraded samples. The quite conserved 5.8S region in fact contains enough phylogenetic signal for discrimination at the level of orders and phyla (29), although identification at this taxonomic level is not the concern of barcoding. Alignments are trivial to optimize for 5.8S due to the few indels found in plants and fungi (30). In contrast for phylogenetic reconstruction, ITS or any rapidly evolving noncoding region can require complex sequence alignment for homology assessments. Thus, the 5.8S locus can serve as a critical alignment-free anchor point for search algorithms that make sequence comparisons for both phylogenetic and barcoding purposes. The utility of conserved regions such as 5.8S to generate a pool of nearest neighbors for refined comparisons will be critical for effective database searches, especially when comparing a sequence that has no identical match in a sequence library. GenBank blast searches with our ITS data (see below) returned correct matches for the sequences in GenBank. This success suggests that despite alignment concerns, current search algorithms will be fast and effective at using ITS for species-level identifications, given an adequate database for comparison. For all of these reasons, ITS, even with its recognized limitations, is a prime candidate as an effective locus for DNA barcoding in plants.

However, the recognition that ITS has certain functional limitations for DNA barcoding of plants is a compelling argument that a search for additional loci is warranted. For phylogenetic investigations, the plastid genome has been more readily exploited than the nuclear genome and may offer for plant barcoding what the mitochondrial genome does for animals. It is a uniparentally inherited, nonrecombining, and, in general, structurally stable genome. Universal primers are available for a number of loci and intergenic spacers that are evolving at a variety of rates. The plastid locus most commonly sequenced by plant systematists for phylogenetic purposes is rbcL, followed by the trnL-F intergenic spacer, matK, ndhF, and atpB (e.g., refs. 31-33). rbcL has been suggested as a candidate for plant barcoding (34), even though it has generally been used to determine evolutionary relationships at the generic level and above. Besides rbcL and atpB, all of the latter plastid loci have been used at the species level with various degrees of success. Most of them (except the trnL-F spacer) require full-length sequences of >1 kb to yield enough sequence length to discriminate species. Most relevant to plant barcoding, no region of the plastid genome has been found to have the high level of variation seen in most animal CO1 barcodes, although a few intergenic spacers have shown more promise than any plastid locus now in general use (33).

When evaluating other genetic loci appropriate for plant DNA barcoding, three criteria must be satisfied: (i) significant species-level genetic variability and divergence, (ii) an appropriately short sequence length so as to facilitate DNA extraction and amplification, and (iii) the presence of conserved flanking sites for developing universal primers. With regard to sequence length, we note that in CO1 barcoding systems, the 600- to 700-bp length fortuitously matches high-quality sequence data from average capillary sequencer reads, although it is expected that routine read length will improve with new technology. An important rationale for using short sequences also resides in the need to obtain useful data from potentially degraded samples found in museum specimens. Amplicon size and gene copy number have been shown to account for much of the variability of amplification success: smaller sizes and increased copy number promote greater success with PCR, presumably by increasing the likelihood that a desired sequence has been preserved (18).

Previous SectionNext SectionMaterials and Methods
Determining Suitable Regions of the Genome. To screen for appropriate levels of sequence divergence in the plastid genome, we chose two closely related flowering plant species for comparison, Atropa belladonna and Nicotiana tabacum (Solanaceae). Both species have complete sequence data available for their plastid genomes (35-37). Twenty-nine additional complete plastid genomes spread across a wide range of plant groups are also available for comparison: algae (five genera in various families), mosses and liverworts (three genera in different families), ferns and relatives (three genera in different families), gymnosperms (two species in the genus Pinus), and angiosperms (eight genera in eight different families, two genera in the Fabaceae, and four genera and several cultivars in the Poaceae). We selected Nicotiana and Atropa, even though they belong to different subfamilies of Solanaceae (38), because they represent the most closely related taxa among the genomes available in the angiosperms. The complete plastid genomes of the taxa in the Fabaceae and the Poaceae include cultivars, hybrids, and more distantly related genera. We aligned the Nicotiana and Atropa genomes, and raw divergence levels (i.e., number of base-pair discordances divided by length of sequence under consideration) were individually estimated across all genes, introns, and intergenic spacers (Fig. 1). Plastid regions with raw sequence differences ≥2% (Table 1) were categorized as the most variable segments, and therefore the most promising of the plastid genome for DNA barcoding when normalized for length. The nuclear ITS region and plastid rbcL gene were used as baseline comparisons for these chloroplast test regions (Table 1). To further narrow down the number of remaining regions usable for barcoding purposes, we applied a sequence criterion of 300-800 bp and a stable presence across multiple plastid genomes of both monocots and dicots.


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Download as PowerPoint SlideFig. 1. Plastid genome variation between deadly nightshade (A. belladonna; shown) and tobacco (N. tabacum), adapted from Shinozaki et al. (35). Shown are a complete genome (A), loci with ≥1% sequence difference between species (B), and loci with ≥2% sequence difference between species (C). The letters in C correspond to spacer regions listed in Table 1.

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In this window In a new windowTable 1. Sampled loci in plastid genomes of Atropa belladonna and Nicotiana tabacum that were found to have base-pair sequence divergences ≥2%
Selecting Taxa for Testing. To empirically test the regions identified as most appropriate for barcoding in our comparison of the plastid genomes of Atropa and Nicotiana (Table 1), we selected two sets of flowering plant taxa. The first taxon set consisted of 2 or 3 species in each of eight genera spread across seven families of plants for a total of 19 species (Table 2 and Table 3, which is published as supporting information on the PNAS web site). The second taxon set included a geographically circumscribed flora comprised of taxa that are not closely related but represent a broad range of angiosperms in 50 plant families, including 83 species in 72 genera (Table 3). The selection of the two taxon sets was made so as to test each locus for appropriate sequence length and divergence, primer success across a wide taxonomic spectrum, and the viability of routinely extracting DNA from dried herbarium specimens, compared with fresh or silica-dried tissue. The species in the first taxon set were selected because they represent a diverse set of species pairs across the angiosperms (including monocots and dicots) with various levels of phylogenetic distance as previously shown in research by the authors using other genetic markers (W.J.K. and K.J.W., unpublished data). In addition, high-quality DNA extractions from living plants, silica-dried tissue, and/or herbarium specimens were readily available for these taxa. The genera were not selected randomly and were not biased a priori toward low or high levels of interspecific divergence. The second taxon set was selected to represent a floristic sample that would be used in a typical plant DNA barcoding project. The samples were taken from Plummers Island, MD, a National Park Service habitat reserve in the Potomac River that has been studied and inventoried by biologists in the Washington, DC, area for >100 years, making it an appropriate test site for barcoding trials. For the Plummers Island taxa, tissue samples were taken from dried leaves only on herbarium specimens located in the U.S. National Herbarium (Smithsonian Institution) collected between 1960 and 2000 (Table 3). These samples were used to compare ITS and rbcL as standards to the best plastid regions identified in the tests of taxon set one. A smaller set of older herbarium collections of Erysimum cheiranthoides (Brassicaceae) prepared as early as 1897 were compared with more recent collections made as recently as 1997 from the same populations to empirically test the relationship between specimen preservation status, age, and DNA quality (see Fig. 2, which is published as supporting information on the PNAS web site).

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In this window In a new windowTable 2. Sequence length and percent sequence divergence for nine plastid regions sampled for eight genera from taxon set one
DNA Analysis. New DNA extractions were performed with the DNeasy Plant Mini kit (Qiagen, Valencia, CA) after tissue disruption of 0.5-1 cm2 of leaf tissue in a FastPrep FP-120 bead mill (Qbiogene, Carlsbad, CA). DNA extractions followed manufacturer's protocols with the modification of buffer AP1 lysis conditions by the addition of 0.4 mg of proteinase, 15 mg of DTT, and incubation at 42°C for 12 h on a rocking platform. This method can easily be scaled up to a 96-well format for large-scale (high-throughput) barcoding purposes. Amplification by PCR used puReTaq Ready-To-Go PCR beads (Amersham Pharmacia Biosciences) and direct sequencing of purified PCR products used bigdye 3.1 software on a 3100 sequencer, both from Applied Biosystems. Universal primers for selected genes and intergenic spacers were taken from investigations described in refs. 39-41 and Table 4, which is published as supporting information on the PNAS web site. Comparative rbcL data were generated for the Plummers Island flora by splitting the gene into two overlapping fragments (1f-724r and 636f-1368r), because test amplifications on a portion of the samples netted only 31% success as a full-length fragment vs. 94% as two pieces.

Previous SectionNext SectionResults
In our comparison of the Atropa and Nicotiana plastid genomes, the most variable regions that tentatively met the barcode criteria were nine intergenic spacers: trnK-rps16, trnH-psbA, rp136-rps8, atpB-rbcL, ycf6-psbM, trnV-atpE, trnC-ycf6, psbM-trnD, and trnL-F (listed in order of decreasing variability; Table 1 and Fig. 1). By comparison, ITS had a much higher divergence value (13.6%) than any of the plastid regions, and rbcL was by far the lowest in divergence (0.83%). Although three spacers (atpB-rbcL, ycf6-psbM, and psbM-trnD) were slightly to moderately longer than our 800-bp cutoff, we included them in our further analysis because of their high interspecific variability.

The results of our intrageneric tests across eight genera in the first taxon set demonstrated conspicuous differences between the nine plastid regions with respect to our three barcoding criteria: amplification success, sequence length, and sequence divergence. Only three regions (trnH-psbA, rp136-rpf8, and trnL-F) were successfully amplified for all eight genera and 19 species; the other regions, including ITS, could not be amplified in one or more taxa (Table 2). Sequence length in the nine plastid regions ranged from 204 to 1,240 bp, with mean length in all but two (ycf6-psbM and psbM-trnD) falling within our 300- to 800-bp optimum length criterion (Table 2). ITS had the highest between-species sequence divergence values in four of the five genera successfully amplified (Table 2), with a mean sequence divergence of 2.81% across the five genera. trnH-psbA ranked first in divergence value in six of the eight genera and in 11 of the 14 species pairs, compared with the other eight plastid regions; trnV-atpE and trnC-ycf6 ranked highest for the remaining two genera and three species pairs (Table 2). trnH-psbA ranked highest (1.24%) in mean percent sequence divergence across all genera, whereas trnV-atpE (0.29%) and ycf6-psbM (0.30%) ranked lowest (Table 2).

In our broader taxonomic sampling of the Plummers Island flora in which only herbarium material was used, none of the loci could be successfully amplified for all of the 83 species tested, which we suggest may be related to primer design or to more fundamental changes in gene structure during herbarium specimen preparation and storage (see ref. 33). Amplification success was highest for trnH-psbA (100%), followed by rbcL (5′ half; 95%), and ITS (88%, although high-quality sequence data were not obtained from all ITS amplifications). We could not detect any general correlation between specimen age and amplification success, indicating that herbarium specimens in apparently good condition and as old as 20 years can be successfully used to establish DNA-sequence reference libraries. Moreover, amplification of full-length ITS was possible (results not shown) for the five specimens of Erysimum cheiranthoides collected between 1897 and 1997 (Fig. 2), indicating that significantly older specimens also may be used.

Because of the high sequence divergence value in the majority of genera in our taxon set one and the high amplification success of the trnH-psbA spacer in all of our test samples, this region became the focus of our examination of the plastid genome for further analyses of barcoding potential. The trnH-psbA amplicon ranged from 247 to 1,221 bp, whereas the intergenic spacer alone (excluding primer-binding regions and small regions of flanking exon) ranged from 119 to 1,094 bp across 53 families of flowering plants, including both the Plummers Island species and the taxonomic groups (extremes were Thalictrum and Trillium, respectively; see Table 2 and Table 5, which is published as supporting information on the PNAS web site). Most taxa (92%) had amplicons falling between 340 and 660 bp, which is within our suggested length criterion for successful barcoding. All species in our sampling had unique trnH-psbA spacer sequences, which is very relevant to the question of using this gene for barcoding plants.

Previous SectionNext SectionDiscussion
The ITS and rbcL loci provide a baseline against which to compare other genes and intergenic spacers in our directed search for sequences to use in plant DNA barcoding. Besides ITS, those single-copy nuclear genes or their introns that are gaining prominence in species-level molecular systematics studies (e.g., leafy, waxy, pistillata, and RPB2), also were considered. However, because of the lack of universal primers (either published or with potential development by using current information) and poor success by using existing primers, these loci have been eliminated as potential barcode loci. The poor success by using existing primers is probably due to the difficulty of amplifying genes with low numbers of copies in degraded samples and the frequent need to clone PCR products before sequencing. We, therefore, turned our attention to the plastid genome in search of the most variable sequences that would also meet the criteria needed for maximum utility (i.e., variability, universal primers, and short length) and that could be used in place of or in addition to the ITS region. The significantly greater length of rbcL (usually 1,428 bp; Tables 1 and 2) causes problems because it is necessary to use four primers for double-stranded sequencing of the entire gene. Although this number of primers is equivalent to that needed if a two-loci system is used for barcoding purposes, the level of interspecific variation we observed in rbcL is less than the variation detected in either ITS or trnH-psbA alone (Table 2). Furthermore, this gene has been previously discounted for discrimination at the species level (e.g., refs. 31, 42, and 43).

We suggest that the trnH-psbA intergenic spacer is the best plastid option for a DNA barcode sequence that has good priming sites, length, and interspecific variation. In our trials across a diverse set of genera in seven plant families, three plastid regions (trnH-psbA, rp136-rpf8, and trnL-F) ranked highest with respect to amplification success and appropriate sequence length, but trnH-psbA demonstrated nearly 3 times the percentage sequence divergence of these other two regions (1.24% in trnH-psbA vs. 0.44% in both rp136-rpf8 and trnL-F; Table 2). The two spacers with the next highest mean sequence divergence after trnH-psbA (atpB-rbcL at 0.63% and trnC-ycf6 at 0.55%) could not be amplified in one or more of the test genera. In only one genus (Solidago; Asteraceae), exceptionally low sequence divergence in trnH-psbA prevented discrimination among the three species tested, although insertion/deletion differences still allowed us to distinguish among the species. This lack of sequence divergence between taxa was true for one or more species pairs in ITS and all other plastid spacers, except atpB-rbcL, in our test sample. In only 2% of our samples did homopolymer regions adversely affect sequence quality in trnH-psbA.

For a number of reasons, we refrained from a statistical test of differences among mean sequence divergences of the nine spacer regions. First, the sample size in our survey was too restricted to provide a meaningful statistical test (although the standard error of the mean of trnH-psbA does not directly overlap with the means of any of the other spacers). More importantly, as pointed out by Shaw et al. (33), genera within and between families of plants are phylogenetically nonequivalent, i.e., lineages recognized as genera may have quite different divergence rates depending on the various life history traits of the included species. Therefore, statistical comparisons between genera with respect to genetic distance are not valid or warranted at this time. Our intent in calculating these mean percent divergences across loci is to provide a qualitative evaluation of each spacer region for barcoding purposes. In this respect, we consider the high divergence value of trnH-psbA, which permits species discrimination in the largest number of taxa we tested (six of the eight genera and 11 of the 14 species pairs), as strong support for its use as a plant barcode.

The universality of trnH-psbA for differentiating among all flowering plant species clearly needs further investigation (see below), especially in taxa with extremely short spacers that may not contain enough sequence variation for species-level discrimination (e.g., Thalictrum and Solidago in our study and Minuartia in ref. 33). This spacer region also is present in other nonflowering land plants. In a search of GenBank, we found that the trnH-psbA spacer has been successfully amplified in angiosperms, gymnosperms, ferns, mosses, and liverworts, although we do not know at this time the degree of between-species divergence. Further study is needed to determine whether this plastid region is as variable in the nonflowering plants as we have shown for our test angiosperms, and therefore whether it is of broad utility as a barcode across the total spectrum of land plants.

Our findings on the properties of trnH-psbA agree with Shaw et al. (33) in their extensive survey of noncoding plastid DNA for phylogenetic purposes. By applying our barcode criteria (i.e., length considerations and universality) to the framework of their study, we conclude that trnH-psbA has greater potential for species-level discrimination than any other locus they analyzed. Similar to our results, they demonstrated that trnH-psbA amplified and sequenced easily with an average length of 465 bp across the 30 taxa they surveyed. Although this region was the second most variable of the 21 spacers they tested in terms of potentially informative characters, they ranked its utility for phylogenetic purposes as low (tier 3) because of its short length. Our analysis of the number of nucleotide substitutions within genera across all taxa in the 21 plastid regions presented by Shaw et al. (33) indicates that the trnH-psbA spacer has the highest percentage nucleotide difference (0.0135 difference per base pair), even though at least 8 of the 21 other regions showed a greater total number of nucleotide substitutions because of their longer length. The interspecific nucleotide differences in trnH-psbA ranged from 18% to 105% higher than that of the other eight most variable plastid regions. Because short sequence length is an important criterion for barcoding, the high frequency of nucleotide differences of trnH-psbA, in combination with its relatively short length, is a significant advantage. Other studies also have shown a high percentage of interspecific divergence for trnH-psbA, and in most cases, the highest in all plastid regions tested (e.g., refs. 44-48).

Despite this high level of interspecific variation, trnH-psbA has found only limited use in species-level phylogenetic reconstruction because of the short length as well as the difficulty of alignments resulting from a high number of indels (e.g., refs. 49-51). In contrast with the problems of indels for phylogenetic construction, we suspect that indels will ultimately enhance the information needed for species identifications, once the appropriate informatics tools for barcoding are developed. In the set of species we sampled, sequences were alignable within genera, but problematic above that rank. In the one case (Solidago) where sequence divergence was not sufficient to separate species, the presence of unique indels allowed easy discrimination among the taxa. Blaxter (34) advocates ease of alignment as a criterion when evaluating the utility of barcode loci. We do not consider difficulty of alignment to be a major obstacle to the applicability of either ITS or trnH-psbA for the primary purpose of DNA barcoding, i.e., identification. Although ease of alignment is desirable, it is not necessary for barcoding. Searches in GenBank by using our data from both loci with a blast search returned correct identities at both the gene and species level. blast searches are anchored and canalized by conserved regions in both loci, 5.8S in ITS and the small region of flanking exon for trnH-psbA. Intraspecific variation in both ITS and trnH-psbA is known to be relatively low, compared with interspecific variation (27, 52), although in the present study, our intraspecific sampling was insufficient to address this issue.

The extraction of DNA from specimens in herbarium collections was highly successful. This success may be due to the specimens having been air-dried and in a good state of preservation as evidenced by the generally green appearance of the leaves selected for extraction (Fig. 2). Plant voucher specimens vary in how and when they are dried after being pressed. If specimen-drying facilities are not immediately available, especially in humid tropical climates, botanists often treat pressed specimens with ethanol to temporarily preserve them against fungal attack and degradation. Alcohol has been shown to be detrimental to recovering high-quality DNA (53), although how it will affect the short sequences needed for barcoding is unknown. We are encouraged by the fact that museum specimens of insects dried from ethanol storage readily yield CO1 sequences. A more thorough investigation and optimization of methods to extract high-quality barcode DNA from herbarium collections in a high-throughput format will be critical to efficiently build a sequence-database library for plant DNA barcodes. Our positive results by using well preserved specimens indicate that the a priori selection of apparently undegraded plant samples will be an important determinant of success. Fortunately, herbaria often have more than one specimen per species among which to select for successful DNA barcoding.

We have shown here that there are gene sequences suitable for DNA barcoding of flowering plants. It may be necessary to employ more than one locus to attain species-level discrimination across all flowering plant species. Algorithms for combining barcoding sequences from two or more DNA regions to yield species-level unique identifiers are now needed. We believe that ITS and trnH-psbA serve as good starting points for large-scale testing of DNA barcoding across a large sample of angiosperms. A good test would be to expand taxon sampling through the application of both ITS and trnH-psbA to barcode the estimated 8,000 species of flowering plants of Costa Rica (54).

Animals as bioreactors-Therapeutic proteins

The first microbial bioreactors, in particular Escherichia coli and Saccharomyces cerevisiae, were found to be satisfactory for the production of simple polypeptides such as insulin and human growth hormone. However, microbial bioreactors were found to be unsuitable for proteins with complex post-translational modifications or intricate folding requirements, such as the coagulation factors, or monoclonal antibodies. This led to the development of large-scale mammalian cell culture, for example, the use of Chinese Hamster Ovary (CHO) cell bioreactors.
These technologies permitted the development of numerous monoclonal antibodies, cytokines, and other complex bioactive biomolecules. However, there are proteins that, due to a combination of complex structure and large therapeutic dosing, have until now eluded recombinant production using traditional bacterial and cell culture bioreactors. For example, commercial recombinant production of complex molecules, such as antithrombin and alpha1-antitrypsin, has not yet been achieved in microbial or mammalian cell derived bioreactors. The only source is human plasma because of the high dose needed.
Even human serum albumin, the therapeutic protein used in largest amounts (>400 tons, worldwide), the use of the recombinant form, produced in Saccharomyces cerevisiae, is limited to excipient applications. These are within the practical production capacity of this system but far too small for high-volume therapeutic indication (volume replacement).
Capital investments in production plants represent a significant portion of the development cost of new recombinant drugs. Also, the inherent risk associated with the regulatory approval process is a stimulus for the development of flexible and inexpensive approaches for the manufacture of therapeutic proteins. Milk-specific production offers a way to lessen the bite.

MILK AS A POSSIBLE PRODUCTION MEDIA
Here is the method to achieve milk-specific recombinant protein production. Fuse an expression vector, comprising a gene that is encoded for the human or humanized target protein with mammary gland-specific regulatory sequences, and then insert into the germline of the selected production species. When integrated, the milk-specific expression construct becomes a dominant genetic characteristic that is inherited by the progeny of the founder animal (Figure 1). This general strategy makes it possible to harness the ability of dairy animal mammary glands to produce large quantities of complex proteins.


Figure 1. Schematic representation of the transgenic production process, using the production of rhAT in the milk of transgenic goats as an example.




Figure 2. Schematic representation of the somatic cell nuclear nuclear transfer process employed for the production of transgenic animals used for the production of recombinant proteins.



SCREENING THE MAMMALS
Transgenic mice have mainly been used for the testing of expression constructs prior to or concomitant with the generation of larger founder transgenic animals. This model allows the relatively inexpensive and rapid evaluation and optimization of transgene constructs and has proven crucial to the development of milk expression technology. The model allows the definition of regulatory sequences that efficiently target expression of heterologous genes to the mammary gland. Obviously, the very limited milk yield from transgenic mice restricts expression of recombinant proteins to small amounts. But this can be sufficient to obtain meaningful data on the protein of interest. As an example, it was possible to purify enough Malaria antigen MSP142 from transgenic mouse milk to test for immune protection in a primate model.
The generation of transgenic rabbits by pronuclear microinjection is straightforward and inexpensive. Relative to ruminants, rabbits have a short gestation interval that allows up to eight lactations per year. However, only 1.5 L of milk can be obtained per lactation, and this limits the value of this expression system to products with a commercial scale in the low-kilogram range; Labor-intensive milking and high husbandry costs could become prohibitive for larger quantities of purified proteins.
Recombinant protein production in the milk of transgenic sows has been reported for human Protein C, factor VIII, and factor IX. Lactating sows can yield a surprising amount of milk (100–200 L) and it has been reported that the porcine mammary gland cells can carry out the complex post-translational modifications (γ-carboxylation, proteolytic processing) on factor IX and Protein C at rates higher than those encountered with mammalian cell and transgenic mouse milk systems.
Transgenic ruminants are obvious candidates for targeting expression of recombinant proteins to the mammary gland. Thousands of years of patient genetic selection have yielded breeds of sheep, goats, and cattle that can produce prodigious quantities of milk. The first published report of production of therapeutic proteins in the milk of transgenic dairy farm animals was the targeting of factor IX and alpha1-antitrypsin to the milk of transgenic ewes.17 Other proteins such as fibrinogen and factor VIII have also been expressed in the mammary gland of transgenic sheep.
Transgenic dairy goats, with an average milk output per doe on the order of 600 to 800 L per natural lactation, have shown to be well adapted to the production of therapeutic proteins. The timeline from initiation of transgene transfer to natural lactation of resulting transgenic does is 16 to 18 months for goats (Figure 3). A large number of production females can be easily generated from a transgenic male using artificial insemination or embryo transfer techniques. Relatively small herds of a few hundred transgenic does can then easily yield several hundred kilograms of purified product per year. This level of production can meet the manufacturing needs of several factors traditionally derived from plasma fractionation and for a large number of recombinant antibodies currently in development.
Dairy cows have a yearly milk output in the range of 10,000 L. Consequently, with concentrations routinely achieved with most mammary gland-specific proteins, yields of tens of kilograms of recombinant proteins can be produced by one lactating transgenic cow. In addition, embryo culture and transfer technologies are well established for cattle breeds, allowing efficient generation of transgenic cows by somatic cell nuclear transfer. However, it takes almost three years from the onset of transgene transfer to obtain milk from a cow's natural lactation. The tremendous scale-up potential offered by transgenic cattle may compensate for this drawback, especially for indications that necessitate large quantities of protein.
ATRYN: THE FIRST TRANSGENICALLY PRODUCED BIOPHARMACEUTICAL
The recombinant production of AT presented numerous challenges. Antithrombin is a complex glycoprotein carrying 4 N-linked glycosylation sites and 3 disulfide bonds. These characteristics, which are crucial for the functions of AT, precluded the use of microbial bioreactors for its recombinant production. In addition, the therapeutic use of AT calls for large amounts, often grams, of purified protein per course of treatment. This ruled out the use of standard mammalian cell culture bioreactors, because production costs with this approach would be prohibitive.
Expression in the milk of transgenic dairy goats was employed. The promoter region of the goat beta-casein gene was linked to hAT cDNA. This transgene was introduced into the chromosomes of goat embryos, which were then transferred to surrogate mothers. The resulting goats carrying this transgene produce the gene product, rhAT, in their milk. Transgenic offspring from the line selected for commercial development consistently express rhAT in their milk at approximately 2 g/L.18 Expression levels of up to 10 g/L were observed in other lines that were not developed further because of timing issues.
The rhAT protein is isolated from the milk of the transgenic females and conventionally purified using tangential flow filtration, heparin affinity chromatography, nanofiltration, anion exchange chromatography, and hydrophobic interaction chromatography, with a yield of greater than 50% (Figure 4). The human AT purified from transgenic goat's milk is structurally indistinguishable from human plasma-derived AT (hpAT) with the exception of the carbohydrates. The main glycosylation differences observed for rhAT were the presence of fucose and GalNAc, a higher level of mannose, and a lower level of galactose and sialic acid. There was also substitution of 40-50% of the N-acetyl neuraminic acid with N-glycolyl-neuraminic acid.18 The terminal sialic acid in the rhAT contained the same 2-6 linkage found in hpAT.
Several independent laboratories have determined that differences in glycosylation of AT do not affect the intrinsic rate constant of the uncatalyzed or heparin catalyzed inhibition of thrombin, indicating that the carbohydrate chains solely affect heparin binding and not heparin activation or proteinase binding functions. Thus, glycosylation does not impact the major biological activity of AT, which is thrombin inhibition, but explains the differences in affinity for heparin and in pharmacokinetics.
The manufacturing process for rhAT has been validated for its viral and prion removal capacity. The rhAT viral validation studies demonstrated that a significant virus reduction of >8.5 to >25.3 log10 (roughly 300 million fold to septillion fold) was accomplished across the distinctly different modes of the rhAT process. All GTC goats are certified free of scrapie in the United States Department of Agriculture (USDA) Scrapie Flock Certification Program and various risk minimization measures have been instituted to protect this highly controlled closed donor goat population. The rhAT purification process has been validated for its ability to reduce scrapie contamination over a 100 billion fold (>11.3 log10 scrapie removal).

Figure 4. Schematic representation of the process used to purify ATryn from the milk of transgenic goats.

Central surveillancesystem for functional facilities of radiochemistry laboratory

Benefits of Virtual Instrumentation-
Less expensive than actual bench top instruments and while replicating all the functions of
the instrument.
Ability to support custom requirements with minimal cost and testing time.
Better connected to PC technology such as analysis capabilities, remote control.
Product Used- LabVIEW 8.2
Challenge- Developing a fully featured, flexible, powerful and reliable Data acquisition
and control system for instruments implemented with Modbus protocol.
Solution- Establishing serial communication between all the instruments and a computer.
Develop an application in LabVIEW real time software which provides user interface to
acquire data from the instruments and supports various other features.
Description of Application-
In Radiochemistry Lab facilities like Gamma Area Monitors to detect
-radiation
leakage, Ventilation System using filters to purify contaminated air etc. are available at
different locations for safe laboratory operation. Common features of all instruments are that
they are implemented with Modbus Protocol which provides internal standard to
microcontroller for parsing messages and contain RS485 serial ports for establishing serial
communication. A typical Gamma Area Monitor displays current Dose rate, Alarm status,
EHT value, Instrument address etc. and filters in Ventilation system measure Air velocity and
Differential pressure of air.
Central surveillance system developed using LabVIEW acquires all these parameters from
instruments on a computer. So from a computer all the instruments can be monitored and
controlled. A database of parameters is maintained on central computer with date and time
stamp for future reference. From central computer readings can be made available on Local
area network and alarms can be put on Public announcement system.
In the core of LabVIEW application is the development of drivers for instruments
implemented with Modbus protocol. Then making appropriate driver calls in other VIs to
collect data from each instrument one by one in a loop as fast as possible, displaying the data
in virtual panels created for all these instruments and updating data at regular intervals.
System Set-up-
Announcement System
Converter
(RS232 RS485)
Central Computer Daisy
chain form
(Labview application running)
Figure 1. System Topography
Central Surveillance System was developed to communicate with all the instruments
implemented with Modbus protocol and having RS485 serial interface. Serial communication
is established between various instruments (which are connected to each other in daisy chain
fashion) and a computer. A RS232  RS485 converter is required to convert RS232 signal
coming from the computer to RS485 signal going to instruments. RS485 standard provides
advantage of communication over longer distances (due to its differential signal which has
immunity to noise), communication at faster rates, connecting several data terminal
equipments (DTEs) in a network structure.
The central computer is connected to other computers in LAN to make use of features of web
publishing & email sending. LabVIEW application continuously runs on this central
computer to continuously acquire data.
Software Implementation-
Modbus protocol defines message structure that microcontroller of instrument will recognize
and use. In this project, it was implemented using asynchronous serial transmission over
RS232 and RS485 media. Microcontrollers were setup to communicate over standard
Modbus network using RTU (Remote Terminal Unit) transmission mode. The transmission
mode in serial communications defines the way Modbus messages are coded. In RTU mode,
each byte in a message contains two 4-bit hexadecimal characters.
Modbus message structure is shown below-
LAN
Instrument drivers were created in LabVIEW using various VISA functions.
VISA Configure Serial Port- Serial Port of computer is configured using parameters
VISA resource name (COM 1) baud rate (19200), data bits (8), stop bit (1), parity (none),
flow control (None), timeout (10 sec).
VISA Write- Sends Modbus message string to serial port of computer.
Modbus message is created using instrument address, function code, register address, No. of
registers and CRC (cyclic redundancy check) values. Instrument address and register address
are user inputs while function code and CRC are generated using these. An algorithm is
implemented in labview to generate CRC. All message fields are then concatenated to form
message string.
VISA Read- After some time delay (say 50 ms) VISA Read reads the message bits
available on serial port. Incoming message string is decoded to obtain desired value.
VISA Close- Finally, VISA session is closed.
For example to obtain Dose rate from Gamma area monitor addressed 100, Modbus message
values will be-
Instrument address- 100; Function- 04; Register address- 30001; No. of Registers- 01; CRC-
56 63
These instrument drivers were then used to create Virtual Panels for instruments. For Gamma
Area Monitor shown below
Figure 2. Gamma Area Monitor
Virtual panel looks like-
Figure 3. Virtual Panel for Gamma Area Monitor
It displays Dose Rate, Alarm Status, EHT value, set Alarm value etc which replicates actual
Gamma Area monitor. If alarm is present, it can be acknowledged from virtual panel itself.
Similarly virtual panels for other instruments can be created using those drivers.
A separate VI was created to display all instruments present in network simultaneously.
Figure 4. Main Panel*
It displays some parameters for each instrument. Detail analyses can be obtained for an
instrument by clicking for that instrument. If some instrument is not working in network, then
display for that instrument can be switched off to save time.
Application works in loop i.e. after acquiring data from instrument 1, it goes to instruments 2
to acquire data and after acquiring data from last instrument in the network, it comes back to
instrument 1. Readings for each instrument are updated in regular intervals of say 1 minute.
Average of 4-5 readings is calculated for each instrument in every 5 minutes and stored in a
file with time and date stamp for future reference.
*Testing was performed for two Gamma Area Monitors. But the concepts can be extended
for more instruments.
For every instrument calibrated readings are obtained. Average and std. deviation are
calculated from calibrated values to obtain 3 range for each instrument. These values are
stored in a reference file. For this job, a separate VI was created-
Figure 5. Configure Instruments Screen**
Here already set calibrated values for each instrument are displayed as soon as this VI is
opened. Reference file address, reading storage file address, total number of instruments
connected and calibrated values for each instrument can be modified here.
**Numeric blocks to enter calibrated readings are shown only for 4 instruments.
To view past readings or do 3 comparison, a separate VI was created.
Figure 6. Past Data Display Screen***
Here reading storage file name is entered to display contents of that file. It displays readings
with time & date stamp. A particular instrument can be selected to plot readings (mean of
readings) for that instrument with time on x-axis along with 3  range.
***A typical graph should look like this. Here readings of both instruments are zero due to
absence of radiations.
What initially available to operator is CSS Option Screen shown belowFigure
7. CSS Option Screen
CSS Option Screen
Configure Panel Main Panel History Panel
GAM-1 GAM-2 ….. Instrument- N
Calibrated Value Dose Rate Past
Readings
No. of Instruments Alarm Status Plot
with 3 range
References file EHT
Reading storage file Set Alarm
Instrument Drivers
LabVIEW 8.2-
As it can be observed, LabVIEW played an important role in entire project. Some general
properties are mentioned below which were useful -
Graphical Programming, which is unique feature of LabVIEW.
Various options (fonts, colors, decorations etc.) available to make front panel more attractive
and user friendly.
LabVIEW points out error by showing broken “run” button and exact location of error can
also be easily found out.
If correct results are not coming, “highlight execution” can be used to see the actual flow of
data in program.
Lots of in-built functions are available which can be directly used in program.
Lots of examples and LabVIEW HELP can be examined to clear doubts.
Some specific properties related to project are mentioned below-
VISA provides simple functions in LabVIEW which can be used to interface with serial port.
Various VI panels can be linked together i.e. control can be transferred from one VI to other
VI.
Web Publishing Tool makes it easy to make application available on LAN. Also using
functions related to sending email are very easy to implement.
Advantages:
Time- This application saves tremendous amount of time. RCL operator needs to sit in front
of central computer to monitor entire lab meanwhile he can perform his other works as well.
If some mishap happens i.e. radiation leakage then alarm comes on central computer within
seconds so immediate action can be taken. Operator can set the instrument parameters also
from computer itself.
If delay of 50ms is given between serial write & read and total number of
instruments connected in network are 16, then total time taken to obtain all readings once is
50ms*6*16 + 1sec(refreshing time) = 5.8 sec (quite small).
Past Readings- A very good feature of this application (which I also like the most) is that it
keeps track of past readings which can be used for future reference. Also various types of
analyses can be performed with these readings for e.g. 3 comparison. It is impossible
without using this application.
Availability on LAN- This application can be made available on LAN. So, higher
authorities of RCL can view status of instruments by giving URL of this application on their
computers. They need not to install LabVIEW on their computers also.
Conclusion:
Project aimed at developing a central surveillance system in RCL for functional facilities
available. The proposed system uses a LabVIEW application running on central computer
which provides GUI to monitor and control the instruments implemented with Modbus
protocol. Serial communication was established between instruments and central computer.
Other features like web publishing, email facility, voice announcement, past data storage
were added to make this application powerful.