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Activity Corner

BIOTECHNOLOGY CROSSWORD PUZZLE

CLUES

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ArticlesBiofuelProductionandSyntheticBiology

Bringing the costs of producing these ad-vanced biofuels down to competitive levelswith petrofuels, however, is a major chal-

lenge. Researchers at the U.S. Department

of Energy (DOE)s Joint BioEnergy Institute(JBEI), a bioenergy research center led by

Berkeley Lab, have taken another step to-wards meeting this challenge with the de-

velopment of a new technique for pre-treat-ing cellulosic biomass with ionic liquids

- salts that are liquids rather than crystals at

room temperature. This new technique re-quires none of the expensive enzymes used

in previous ionic liquid pretreatments, andmakes it easier to recover fuel sugars and

recycle the ionic liquid.Most of our ionic liquid efforts at JBEI have

focused on using enzymes to liberate fer-

mentable sugars from lignocellulosic bio-mass after pretreatment, but with this new

enzyme-free approach we use an acid asthe catalyst for hydrolyzing biomass poly-

saccharides into a solution containing fer-mentable sugars, says Blake Simmons, a

chemical engineer who heads JBEIs De-

construction Division and was the leader ofthis research. Were then able to separate

the pretreatment solution into two phases, asugarrich

water phase for recovery and a lignin-richionic liquid phase for recycling. As an add-

edbonus, our new pretreatment technique

uses a lot less water than previous pretreat-ments.

With the burning of fossil fuels continuing toadd 9 billion metric tons of excess carbon

dioxide to the atmosphere each year, theneed for carbon neutral, cost-competitive

renewable alternative fuels has never been

greater. Advanced biofuels, produced from

the microbial fermentations of sugars in lig-nocellulosic biomass, could displace gaso-line, diesel and jet fuel on a gallon-for-gallon

basis and be directly dropped into todaysengines and infrastructures without impact-

ing performance. If done correctly, the use

of advanced biofuels would not add excesscarbon to the atmosphere. Environmental-

ly benign ionic liquids are used as greenchemistry substitutes for volatile organic

solvents. While showing great potential as abiomass pretreatment for dissolving

lignocellulose and helping to hydrolyze the

resulting aqueous solution into fuel sugars,

the best of these ionic liquids so far haverequired the use of expensive enzymes.Recent studies have shown that acid cata-

lysts, such as hydrochloric or Brnsted, caneffectively replace enzyme-based hydroly-

sis, but the subsequent separation of sug-

ars and ionic liquids becomes a difficult and

expensive problem can require the use ofsignificant amounts of water.Guided by molecular dynamics simula-

tions carried out at DOEs National Ener-

gy Research Scientific Computing Center(NERSC), Simmons and his colleagues at

JBEI solved this problem by deploying theionic liquid imidazolium chloride in tandem

with an acid catalyst as Imidazolium is themost effective known ionic liquid for break-

ing down lignocellulose and the chloride

anion is amenable with the acid catalyst.The combination makes it easy to extract

fermentable sugars that have been liberat-ed from biomass and also easy to recover

the ionic liquid for recycling. By eliminatingthe need for enzymes and decreasing the

water consumption requirements of more

traditional ionic liquid pretreatments weshould be able to reduce the costs of sug-

ar production from lignocellulose. Completeseparation of the pretreatment solution into

sugar-rich water and lignin-rich ionic liquidphases was attained with the addition to the

solution of sodium hydroxide.

The production of commercially attractivebiofuels using enzymatic methods, all the

same, is not as easy as it appears. The var-ious polysaccharides viz. cellulose, starch,

lignin, hemicellulose, or lignocellulosesneed to be enzymatically degraded for their

transformation into glucose or sugar mole-

cules which in turn are fermented into biofu-els (bioethanol or biobutanol). In case of cel-

lulose, the process of cellulolysis involvesenzymes like cellulases and glucosidases.

Cellulases are expensive, unstable and slowin action; therefore they increase the overall

economics of the process of cellulolysis and

hence biofuel production. The bulk produc-

tion of cellulases at industrial level seemsto be the relevant solution. The microbesthat produce cellulases include symbiotic

anaerobic bacteria (e.g. Cellulomonasfimi,Clostridium thermocellum, Clostridium phy-

tofermentans, Thermobifidafusca ) found in

ruminants such ascow and sheep, flagellate protozoa present

in hindguts of termites, and filamentous fun-gi isolated from decaying plants (e.g. Hypo-

creajecorina, Thermoascusaurantiacus,Phanerochaetechrysosporium, Neurospo-

racrassa, Tricodermareesei, Asperigillus-

niger, Fusariumoxysporum). The gene(s)

responsible for cellulase production arecharacterized, isolated and recombinantlyintroduced into Escherichia coli for the en-

hancedcellulase expression levels. Apartfrom the conventional biotechnology meth-

ods for biofuel production, synthetic biology

has shown promising results lately.

Understanding the DNA sequences, pre-

cisely measuring the gene behaviour pavesway for fabricating or synthesizing the cellu-

lase gene de novo. To put it in simple words,synthetic biology is a science of designing

and constructing new biological parts, de-vices and systems for programming cells

and organisms and endowing them with

novel functions. It is a technique of writingthe DNA / genetic code base by base using

several computational tools and softwareslike Gene designer, GenoCAD, Eugene and

Athena to name a few. Gene designer is aDNA design tool for de novo assembly of

genetic constructs, GenoCAD is a comput-

er-assisted-design application for syntheticbiology for designing complex gene con-

structs and artificial gene networks, Eugeneis a language designed to develop novel

biological devices and Athena is a CAD /CAM software for constructing biological

models asmodules. These synthetic biology

approaches can be useful in bringing down

the cost of cellulases and, thereby, of biofu-els. Several companies are spending a for-tune on the production of bioethanol for ex-

ample; Amyris Biotechnologies, Verenium,Iogen, Bioethanol Japan, Mascoma, POET,

SolixBiofuels, Pacific Ethanol, NextGen

Fuel Inc. and Jatro Diesel. However, thecost-effective production of the second gen-

eration biofuels is still a cherished desire ofthe scientific community. Synthetic biology

is an evolving field still dealing with the in-herent complexity of biological systems and

overcoming the biosafety issues involved

with engineering the living systems. Indeedthe proliferation of the computer modelling

tools is leading to the revolution of this dis-cipline which might write the success story

of some of the present and future scientificchallenges.

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BiodegradableBattery:Thebat-terythatmeltsinsidethebody!

A four-cell battery made of biodegradablematerials completely dissolves after three

weeks in water.

Such biodegradable, implantable batterycould help in the development of biomedical

devices that monitor tissue or deliver treat-ments before being reabsorbed by the body

after use. This is a really major advance,

says Jeffrey Borenstein, a biomedical en-gineer at Draper Laboratory, a non-profit

research and development center in Cam-bridge, Massachusetts. Until recently, there

has not been a lot of progress in this area.In 2012, materials scientist John Rogers at

the University of Illinois at Urbana-Cham-

paign unveiled a range of biodegradable sil-icon chips that could monitor temperature

or mechanical strain, radio the results toexternal devices, and even heat up tissue

to prevent infection. Some of those chips re-lied on induction coils to draw wireless pow-

er from an external source.

But wireless power transfer is problemat-ic for devices that need to go deep within

tissue or under bone. The components thatreceive the power are also quite complex,

anything put in is going to take space.. Toprovide a tidier solution, Rogers and his col-

laborators have now created a fully biode-gradable battery.

Dissolvable devicesThese devices use anodes of magnesium

foil and cathodes of iron, molybdenum ortungsten. All these metals slowly dissolve in

the body, and their ions are biocompatible in

low concentrations.The electrolyte between the two electrodes

is a phosphate-buffered saline solution,and the whole system is packed up in a

biodegradable polymer known as a poly-anhydride. Currents and voltages vary de-

pending on the metal used in the cathode.

A one-square-centimeter cell with a 50-mi-crometer-thick magnesium anode and an

8-micrometer-thick molybdenum cathodeproduces a steady 2.4 milliamps of current,

for example.

Once dissolved, the battery releases less

than 9 milligrams of magnesium roughlytwice as much as a magnesium coronary

artery stent that has been successfullytested in clinical trials, and a concentra-

tion that is unlikely to cause problems inthe body, says Rogers. Almost all of the

key building blocks are now available toproduce self-powered, biodegradable im-

plants, he says.

All versions can maintain a steady output

for more than a day, but not much longer.The team hopes to improve the batteries

power per unit weight known as pow-er density by patterning the surface of

the magnesium foil to increase its surfacearea, which should enhance its reactivity.

The authors estimate that a battery mea-

suring 0.25 cm2 and just one micrometerthick could realistically power a wireless

implantable sensor for a day.

In the fieldThe devices could also find environmen-

tal applications, says Borenstein. For ex-

ample, to help remediation efforts duringan oil spill, environmental officials could

drop hundreds of thousands of tiny wire-less chemical sensors across the slick.

These would later simply dissolve inthe ocean. Space is less of a constraint

in these applications: a stack of severalcells, for instance, can produce up to 1.6volts enough to power a light-emitting

diode or generate a radio signal.

Magnesium batteries are not the onlysolution. Last year, biomaterials scientist

Christopher Bettinger of Carnegie Mellon

University in Pittsburgh, Pennsylvania,unveiled an edible sodium-ion battery

with electrodes made from melanin pig-ments. But Rogers team report that their

magnesium batteries have a relativelyhigher current and power density, and

last for longer.

Borenstein hopes that further research

into both types of batteries could even-tually yield implantable drug-delivery de-

vices that are controlled by radio signals,or that dispense pharmaceuticals in re-

sponse to a specific acute problem, such

as an epileptic seizure.

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LightActivatedNeuronsFromStemCellsRestoreFunctionstoParalyzedMuscles

The Concept:

A new way to artificially control muscles us-ing light, with the potential to restore func-

tion to muscles paralyzed by conditionssuch as motor neuron disease and spinal

cord injury, has been developed by scien-tists. The technique involves transplanting

specially designed motor neurons created

from stem cells into injured nerve branches.These motor neurons are designed to react

to pulses of blue light, allowing scientists tofinetune muscle control by adjusting the in-

tensity, duration and frequency of the lightpulses.

The Technique:The technique involves transplanting spe-

cially designed motor neurons createdfrom stem cells into injured nerve branch-

es. These motor neurons are designed toreact to pulses of blue light, allowing scien-

tists to finetune muscle control by adjusting

the intensity, duration and frequency of thelight pulses.

Following the new procedure, we saw pre-viously paralyzed leg muscles start to func-

tion, says Professor Linda Greensmith ofthe MRC Centre for Neuromuscular Dis-

eases at UCLs Institute of Neurology, who

coled the study. This strategy has signifi-cant advantages over existing techniques

that use electricity to stimulate nerves,which can be painful and often results in

rapid muscle fatigue. Moreover, if the exist-ing motor neurons are lost due to injury or

disease, electrical stimulation of nerves isrendered useless as these too are lost.

Muscles are normally controlled by motor

neurons, specialized nerve cells within thebrain and spinal cord. These neurons relay

signals from the brain to muscles to bringabout motor functions such as walking,

standing and even breathing. However, mo-tor neurons can become damaged in mo-

tor neuron disease or following spinal cord

injuries, causing permanent loss of musclefunction resulting in paralysis.

This new technique represents a meansto restore the function of specific muscles

following paralysing neurological injuries ordisease, explains Professor Greensmith.Within the next five years or so, we hope to

undertake the steps that are necessary totake this groundbreaking approach into hu-

man trials, potentially to develop treatmentsfor patients with motor neuron disease,

many of whom eventually lose the ability to

breathe, as their diaphragm muscles gradu-ally become paralyzed. We eventually hope

to use our method to create a sort of opticalpacemaker for the diaphragm to keep these

patients breathing.We custom-tailored embryonic stem cells

so that motor neurons derived from them

can function as part of the muscle pace-maker device. says Dr Lieberam, who coled

the study. First, we equipped the cells witha molecular light sensor. This enables us to

control motor neurons with blue light flash-es. We then built a survival gene into them,

which helps the stemcell motor neurons to

stay alive when they are transplanted insidethe injured nerve and allows them to grow

to connect to muscle.

The Concept:Living skeletal muscle that contracts pow-

erfully and rapidly, integrates quickly intomice, and for the first time, demonstrates

the ability to heal itself both inside the lab-oratory and inside an animal has been

grown in the lab by biomedical engineers.

The muscle we have made represents animportant advance for the field, an author

said. Its the first time engineered musclehas been created that contracts as strongly

as native neonatal skeletal muscle.The Technique:

The study conducted at Duke University

tested the bioengineered muscle by literallywatching it through a window on the back of

living mouse. The novel technique allowedfor realtime monitoring of the muscles in-

tegration and maturation inside a living,walking animal. Both the labgrown muscle

and experimental techniques are import-ant steps toward growing viable muscle for

studying diseases and treating injuries, said

Nenad Bursac, associate professor of Bio-medical Engineering at Duke. Every muscle

has satellite cells on reserve, ready to acti-vate upon injury and begin the

regeneration process. The key to the teamssuccess was successfully creating the mi-

croenvironments called niches where these

stem cells await their call to duty. Simplyimplanting satellite cells or lessdeveloped

muscle doesnt work as well, said Juhas.The well-developed muscle we made pro-

vides niches for satellite cells to live in, and,

when needed, to restore the robust muscu-lature and its function.

How the trials took place:To put their muscle to the test, the engineers

ran it through a gauntlet of trials in the lab-oratory. By stimulating it with electric puls-

es, they measured its contractile strength,

showing that it was more than 10 times stron-ger than any previous engineered muscles.

They damaged it with a toxin found in snakevenom to prove that the satellite cells could

activate, multiply and successfully heal theinjured muscle fibers.

Then they moved it out of a dish and into

a mouse. With the help of Greg Palmer, anassistant professor of Radiation Oncology

in the Duke University School of Medicine,the team inserted their labgrown muscle

into a small chamber placed on the backsof live mice. The chamber was then covered

by a glass panel. Every two days for two

weeks, Juhas imaged the implanted mus-cles through the window to check on their

progress.

By genetically modifying the muscle fibers

to produce fluorescent flashes during calci-

um spikes which cause muscle to contractthe researchers could watch the flashes be-

come brighter as the muscle grew stronger.We could see and measure in real time

how blood vessels grew into the implantedmuscle fibers, maturing toward equaling the

strength of its native counterpart, said Ju-

has.The engineers are now beginning work to

see if their biomimetic muscle can be usedto repair actual muscle injuries and disease.

Can it vascularize, innervate and repair thedamaged muscles function? asked Bursac.

That is what we will be working on for the

next several years.

Selfhealingengineeredmusclesgrowninlaboratory

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focusIN

optogeneticssee it, without actually seeing it

When you think of the brain whatdo you see; a group of identicalneurons packed together form-

ing different regions of the brainright?? Well you are wrong, thereare many distinct types of neurons,which are distributed in a highlyorganised fashion in different brainregions and interconnected withexquisite specificity. And when Isay many, I mean many hundreds,possibly thousands of types.

Now imagine a way you could ex-cite a single type, just to see its ef-fect on the organism in real time,welcome to the world of OPTO-GENETICS.

Introduction:Optogentics is a new method ofneural modulation that can even beapplied to freely moving animals.Strictly speaking, optogenetics in-

volves experimenting with a com-bination of genetic manipulationand optics It can be used in sev-eral animal models, including theC. elegans, fly, zebrafish, mouse,rat, and primate .Ultimately, op-togenetics may be used to con-trol the behavior of freely moving

mammals by administering light. Acomplicated explanation wouldnt

you say, so lets break it down.

The first key component of this

technology (I use technology inthe more liberal sense) are opsin, these are light sensitive G-cou-pled proteins ( transmemberaneproteins that are capable of con-verting a stimulus into a cellular re-sponse) . There are broadly of twotypes Type I opsins are employedby prokaryotes, whereas animalsuse Type II opsins. Each opsin type

reacts to a certain wavelength in aunique manner. This allows themto be used for not just activationbut for inhibition as well. Now cellwith these

proteins react by opening a chan-nel in their cell membrane to allowelectrochemical ions (like sodi-um or chloride) to flow in or out ofthe cell when they are subjectedto light of a light of certain wave-

lengths. Controlling the flow ofsuch ions along their fibres is alsohow neurons conduct electricity.

The next step is to make sure thatmammalian cells express microbi-al opsins. Because simply admin-istering a protein will not work, a

gene that encodes for the opsinneeds to be introduced to specif-

ic cells instead. For this to happenwe need to use various methodsof recombinant DNA technology to

facilitate said transfer of genes.One possibility is to use a vector,for example inject a (harmless)virus to carry the opsin gene intothe brain of a mammal. The majordrawback of viral expression sys-tems is that they cannot carry largeamounts of genetic material. How-ever, the advantage is that opsinsare expressed in high levels. An-

other way to introduce opsins is touse transgenic (knock-in) animalsthat possess the opsins from bir th.This obviously has the advantageof studying the development of asystem. However, transgenic ani-mals show lower opsin expressionlevel. Other methods of transfer in-

clude the use of Cre-Lox recombi-nase system, a widely used meth-

UseofprogrammableLCDphotomaskforneuronstimulation

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od to make changes into the DNAat specific locations. Now if youwere to stimulate a particular sitein the brain of the animal youwould see that only a specific typeof neurons that express the opsinfire, ie are excited or activated orare inhibited. This depends on thetype of opsin used. For example

blue light makes channelrhodop-sin-2 (ChR2) rapidly depolarize aneuron.

Now why would we go throughall this trouble? I am sure you arewondering whether there are anyexisting ways of checking neural

response, well there are but manyare nowhere near as specific andsome are downright weird (for ex-ample deep brain stimulation in-volves placing a device in the brainwhich sends electrical impulses tospecific parts of the brain, it is used

for treating diseases like Parkin-son`s )optogenetics provides onemeans to integrate analyses atvery different levels, uniting what

have been disparate areas of neu-roscience.The characteristics of individualneurons or specific synaptic con-nections are traditionally analysedby molecular and cellular neuro-science and electrophysiology.

The roles of specific neurotrans-mitters or receptors are probed

with pharmacology. The functionsand interactions of brain areasare studied using field recordings,electroencephalography, neuroim-aging, lesions and other systemsneuroscience methods.

The advantage of optogenet-ics over other neuromodulationtechniques is its high-temporalspecificity combined with cellularprecision. For example, althoughelectrical manipulation has ahigh temporal resolution (precisetime dependent measurements),it is unable to achieve true inac-tivation or excitation of individu-

al neurons. Pharmacological andgenetic manipulations show theopposite pattern, they can targetat least certain kinds or families ofneurons but are lacking temporal

precision. Thus by allowing us tobetter understand the functioningof various neuron types, their in-teractions with each other and theeffect of pharmacological agentson them; optogenetics gives us a

far more holistic approach to thestudy of the brain that too is a veryefficient manner.

Optogenetics as an importantresearch tool:Relationship between dopamine(DA) neuron firing and positive

reinforcement in genetically mod-ified rats. The use of optogeneticscan lead to a better understandingof cause-effect relationships, forexample in dopamine-based dis-orders such as Parkinson`s.Understanding of sleep patterns.Optogenetics is important for con-trolling neural circuits to examineboundaries between sleep andwakefulness.Several studies used optogenet-

ics to investigate neural circuitsthat underlie fear conditioning andmemory formation, these play animportant role in theunderstandingof fundamental cognitive process-es like memory formation, but also

in anxiety disorders and post-trau-matic stress disorder, which arecharacterized by disturbing, recur-ring contextual memories.Optogenetics has further been ap-plied to cortical oscillations (syn-chronized neural activity), that areassociated with various cognitiveprocesses as well as psychiatricconditions such as anxiety, autism,

and schizophrenia.Conclusion:In all Optogenetics provides us atool to study a myriad set of dis-eases and disorders, with a level

of specificity that was previouslynot possible to achieve. It gives usthe chance to combat diseasesand disorders that were previouslythought untreatable, not to men-tion greater insight into the func-

tioning of the most mysterious andimportant organ of our bodies.Some of the most important workin the field was done in 2005, byresearchers at Karl Deisserothslaboratory first demonstrated asingle-component optogeneticsystem and in 2006 the term op-togenetics was born.

Research in this field is being car-ried out in many of the top univer-sities in the world. For instance,optogenetic modulation in primateneurons has been investigated andit has already been demonstratedthat ChR2 can function within hu-man neurons.

SchematicdiagramofLight-basedstimulation

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EDITORS Creative Team

Kanwar abhay singh Ashima Aggarwal gagan bhasin Ujjal Didar Singh Pujit Singh