Young Investigators Spring 2010 Issue

Young Investigators Review

The Stony Brook

Lyme DiseaseStony Brook Researchers Work Towards a Cure

Writing With LightStony Brook BME Develops a New Micropatterning Technique

Stem Cells A new Therapeutic Hope

Spring 2010Volume 2, Issue 2

Revamping the Premedical CurriculumThe AAMC Proposes Changes

AN UNDERGRADUATE JOURNAL OF SCIENCE

Reviews

2 The Stony Brook Young Investigators Review, Spring 2010

Sponsorship

Funding and Support Provided by: The Stony Brook College of Arts and Sciences The Stony Brook Foundation The Department of Biochemistry and Cell Biology 7KH2IFHRI5HVHDUFKDW6WRQ\%URRNOSI Pharmaceuticals Inc. New England Biolabs Inc. Half Hollow Hills School District 9:5,QWHUQDWLRQDO,QF

Contents

3The Stony Brook Young Investigators Review, Spring 2010

On the Cover6WHPFHOOVDUHXQVSHFLDOL]HGFHOOVWKDWDUHUHVSRQVLEOHIRUSURGXFLQJDOOWKHRUJDQVDQGWLVVXHVLQWKHKXPDQERG\7KH\SURYLGHHQRUPRXVKRSHWRWKRVHVXIIHULQJIURPGHYDVWDWLQJDLOPHQWVOLNH3DUNLQ-VRQV'LVHDVHDQG$O]KHLPHUV'LVHDVH,QWKLVLVVXH

Reviews


Presidents Message!e cardinal aim of our undergradu-

ate enterprise, the Stony Brook Young Investigators Review, is to establish an environment in which students can in-teract with their colleagues and faculty members on a number of di"erent levels, in order to receive instructive feedback on their research projects. !rough print and online publications, and through sympo-siums, such as the Second Annual Young Investigators Symposium, we hope to at-tract students from a number of diverse academic and social back-grounds and to those who have not yet ventured into the world of laboratory work, o"er a warm welcome to the scienti#c com-munity.

As President, it has been an absolute honor to set the stage and showcase the talent and potential we have here at Stony Brook University. I hope that this journal continues to $ourish and will foster an environment which will help continuously expose stu-dents to the ongoing research performed both inside and outside our Universitys grounds. We hope to accomplish this goal by pub-lishing the journal once every semester, increasing the number of copies published, expanding the scope of the material covered to the physical sciences, and to allow for more submissions. Recent-ly, we have established an Internet presence, our website, http://younginvestigators.com, and will be persistently updating it to

include the most recent content. Of course, this will take a great amount of time. However, I hope that the steps we have taken so far have established a strong foundation upon which our next undergraduate leaders can build.

Our latest mission, has been to establish a network for Stony Brook University, which will allow for easier identi#cation of labs which will suit student interest as well as will help faculty mem-bers easily identify quali#ed undergraduates to enroll and assist in accomplishing their research goals. We hope to satisfy our ob-jective by creating an online network interface. We have already entered the planning phase of this project.

I would like to thank Dr. Robert Weinberg for spending his day with Stony Brook University students and faculty members, and for his exceptional keynote address. I would also like to thank Dean Jerrold Stein, Dr. Robert Haltiwanger, Dr. Paul Bingham, Dr. Paul Bynum, Dr. Wali Karzai, Dr. Sanford Simon, the help-ing hands of the Student Activities Center, our writers, and our sponsors, for all of your support, for helping us transform the idea of having a large-scale symposium into a reality and for making the undergraduate collaboration you are holding in your hands a possibility. Additionally, I would like to thank Dr. Michael Lake of the Biochemistry Department, who extended the entire under-graduate research experience to his high school students.

Respectfully Yours,

Isaiah P. SchusterB.S. Pharmacology (2010)PresidentE-mail: [email protected]

5

Reviews

The Stony Brook Young Investigators Review, Spring 2010

You are following some of the bright-est minds in the world.

Indeed, Stony Brook University has no shortage of undergraduates with academic talent and intellectual prowess; a fact that is highlighted on the back of every bus on campus. A great number of these out-standing students can be found within the biological sciences. However, within these departments, there is a large de#cit of stu-dents interested in pursuing graduate study. !e vast majority of Stony Brook Biology and Biochemistry majors have one un$inching goal in common: to gain entrance to medical school. I believe this de#cit in students interested in pursuing a graduate education exists due to a fundamental lack of knowledge; that is, of all that graduate school and Ph.D. programs, in particular, have to o"er bright students.

!e majority, if not all, Ph.D. programs in the natural and applied sciences are fully funded; meaning that tuition is paid for and a living stipend of approximately 30,000 dollars per year is typically provided along with health insurance. !is means that a student will graduate without debt, unlike many profes-sional schools. Perhaps the greatest bene#t of attaining a Ph.D. is that it opens up a host of career opportunities. !is includes academic research, in the form of a faculty position which in-cludes the bene#ts of tenure and teaching, or a career in the biotech industry, which could include your own start-up com-pany if you are particularly entrepreneurial in nature. Gradu-ates of Ph.D. programs have gone on to careers in patent law, public policy, and even scienti#c publication. !e Doctorate of Philosophy is the highest degree that an academic institution can bestow and, as Dean Lawrence Martin often relates, the word doctor is derived from the Latin doctoris, which means teacher. So professors and other holders of the Ph.D. degree are the true doctors.

So why choose a career in research? Because research is ex-citing; the particular scienti#c problem or question that you are trying to address is constantly changing and evolving. Simply put, the job is never the same from one day to the next. One important aspect of science that never seems to be emphasized enough is creativity. By conducting research you get to $ex your creative muscles by designing simple, yet elegant experiments that elucidate information about the inherently complex, natu-ral puzzle under question. Science is also rewarding. By under-taking this endeavor, you are able to expand the human knowl-edge base on how the natural world functions. !e career itself is multifaceted; there is not only bench work in the laboratory but also traveling and presenting at scienti#c meetings, writing papers, and signi#cant mentorship and teaching opportunities.

Being involved with !e Stony Brook Young Investigators Review can prepare you for a career in science in a number of

Letter from the Editorways. Sta" members often write review articles, which require the interpretation and evaluation of papers in the primary lit-erature. Undergraduate contributors can also choose to publish their own independent research #ndings in our journal, which is an important step for any scientist. In fact, we have two in-dependent research submissions in this issue alone and plan on having more in subsequent issues. !e greatest bene#t of work-ing for the Young Investigators Review is that it can get you excited about science; working with like-minded individuals to produce an issue that contains articles on the most cutting-edge research.

Stony Brook certainly has a history of producing strong candidates for graduate study and many graduate school bound undergraduates have gone on to Ph.D. programs at prestigious universities; including Kevin McCarthy, class of 08, who is cur-rently in the Virology Program at Harvard University, Jean-Luc Chaubard, class of 09, who is currently in the Chemistry Department at the California Institute of Technology, Allison Goldberg, class of 10, who is joining the Department of Phar-macology at Yale University in the Fall, and I myself am joining the Department of Biology at the Massachusetts Institute of Technology in September.

It is true that the pursuit of a Ph.D. may not be for ev-eryone, as it requires a signi#cant amount of dedication (aver-age time to completion is about 5.5 years) and a sharp, incisive, and questioning mind. However, it is certainly a career path that promising undergraduates should consider at least once in their academic career. Conducting research and participating in our undergraduate journal of science are two great ways to gain exposure to the world of basic science and to help you, the student, decide if professional school is for you, or if maybe you have a previously undiscovered enthusiasm for science that will lead you to graduate study.

Sincerely,

Kevin KnockenhauerB.S. Biochemistry (2010)Editor-in-ChiefE-mail: [email protected]

Articles


M3: Maskless Microscope-based Micropatterning6WHYHQ/HLJK$DVKD\7DWWX6\HG=DPDQ0LFKDHO%XGDVVL=KLKHQJ-LD+DUROG%LHQDQG(PLOLD(QWFKHYD

Department of Biomedical Engineering Stony Brook University, Stony Brook, NY 11794-8181

Summary

We have developed a new micropat-terning technique using an inverted $uo-rescence microscope with programmable XY stage and layered dry-#lm photore-sist, thermally bound to a glass slide. !e method allows for $exible in-house mask-less photolithography without a dedicated microfabrication facility, and is well suited for in-house fabrication of micro$uidic channels, sca"old templates for protein/cell patterning or optically-guided cell encap-sulation for biomedical applications.

Description of Design Objective

Using an approach inspired by recent work [1] in maskless photolithography, the goal was to develop a new basic photolitho-

graphic technology that is suitable for im-plementation in any biomedical laboratory equipped with an inverted $uorescence-en-abled microscope and programmable stage.

!e design involves a method for trans-lating a desired image into programming commands to the microscope stage as well as investigating the limits of photoresist micropatterning via standard microscope objectives without additional components, e.g. shutters.

Description, Analysis and Performance Evaluation

We decided to use dry-#lm photoresist Ordyl SY 330 (ElgaEurope, Italy) because of its potential to create relatively high-depth features (30m for a single layer). Glass slides were used as a support surface and a standard o%ce laminator was em-

ployed to deposit layers of the photoresist.User-provided images are transferred

onto the photoresist by epi-$uorescent UV light through standard microscope objec-tive lenses. Images are translated into mi-croscope XY-stage movement by custom software written in C++. Speci#cally, we used a 40x Fluor objective lens on a Nikon Eclipse TE2000-U (Nikon, Japan) micro-scope equipped with a Prior OptiScanII programmable stage (Prior Scienti#c, UK), where UV radiation was provided by a 365nm bandpass #lter (Chroma, USA) ap-plied to a broadband xenon arc lamp light source (Cairn, UK). !e illuminated area produces a negative point in the photoresist after etching and baking. In-plane features depend on the objective parameters and the encoded speed, while the height is propor-tional to the thickness of the photoresist layers (30m each). Our results indicate feature dimensions to be commensurate with cell dimensions and acceptable for biological applications. Even though the process is serial in nature, a complex shape can be transferred within minutes. Con-nected patterns (images with one compo-nent) are drawn in a straightforward way with continuous movement of the stage, while disconnected patterns require fur-ther considerations (as discussed below). !e #nal product is a cast made from the photoresist template using biocompatible

)HDWXUH:LGWKYV6WDJH6SHHG5HODWLYH,QWHQVLW\ BA

C

Figure 1. (A))HDWXUHZLGWKDORQJOLQHDUFRPSRQHQWVRISKRWRUHVLVWSDWWHUQVDVDIXQFWLRQRIVWDJHVSHHGPLFURQVSHUVHFRQGDQGUHODWLYHLQWHQVLW\ZKHQXVLQJWKHIXOODSHUWXUHRIWKHREMHFWLYHOHQV1HXWUDOGHQVLW\OWHUVDWWHQXDWHGDIUDFWLRQRIWKHV\VWHPVPD[L-PXP89LQWHQVLW\(B))HDWXUHZLGWKGURSVVLJQLFDQWO\ZKHQXVLQJDSLQKROHDSHUWXUHWRUHGXFHWKHHIIHFWLYH1$RIWKHREMHFWLYHOHQV(C)$Q$UFKLPHGHDQVSLUDODVSDWWHUQHGRQD[PPJODVVFRYHUVOLGH

7

Articles


materials, e.g. polydimethylsiloxane, or a photopolymerized cell-encapsulating gel, etc. Connected Patterns: Full Aperture !e high light-delivering power of the Nikon objective lens used (40x, Plan Fluor DIC M; NA = 0.75) is capable of achieving feature sizes between 250m and 700m. Tuning of feature sizes is possible by careful manipulation of UV dose via neutral den-sity #lters and stage speed (Figure1A).

Connected Patterns: Pinhole Aperture

Applications requiring feature sizes between 40m and 250m are achieved by placing a pinhole aperture behind the objective, reducing the e"ective numerical aperture (NA) of the high-magni#cation lens while preserving its high magni#ca-tion (Figure 1B).

Disconnected Patterns

We have avoided the use of a shutter in this design for simplicity and a"ordability. By taking advantage of results from com-putational geometry and graph theory, we can broaden the systems use to allow for the creation of disconnected patterns. Since the photoresist shows a threshold-like re-sponse to UV radiation $ux integrated over time, two exposures at one location with a dose of [(1+delta)ET]/2 will successfully create an isolated point only at this loca-tion, where ET is the exposure threshold or minimum required amount of delivered UV and delta is an arbitrarily small number to ensure exposure saturation. With careful navigation of the photoresist (via Traveling Salesperson heuristics [2,3] and 2-opt im-provements [4], we prove that it is possible to use this approach to create disconnected patterns.

Discussion

We have demonstrated maskless mi-cropatterning to be possible in a biological laboratory equipped with a standard $uo-rescence microscope system. By controlling the numerical aperture, stage movement speed and the intensity of the delivered light, one can realize a wide range of feature sizes (40m-700m). Feature depth is con-trolled by varying the number of layers of the dry #lm photoresist. Although the cur-rent state of the invention is best suited for connected patterns, a shutter-based design or exposure-based traversing can create any arbitrary pattern.

Acknowledgements

We acknowledge Nicolas Glade (Com-missariat lnergie Atomique, Grenoble, France) for assistance with the methodol-ogy for #lm exposure and developing and ElgaEurope for the donation of the Ordyl SY330 dry #lm resist. We also acknowledge Prof. Joseph Mitchell and Prof. Estie Ar-kin for their advice on the computational geometry and graph theory aspects of the system.

5HIHUHQFHV

1. Breadmore MC, Guijt RM. Maskless photolithography using UV LEDs. !e Royal Society of Chemistry 8 (2008), p. 1402 1404.2. Mitchell JSB and Arkin E, Professors of Applied Mathematics and Statistics. Stony Brook University. Stony Brook, NY. Personal communication. April 28, 2009.3. Tucker, Alan. Applied Combinatorics. San Francisco: Wiley, 2001.4. Lin S and Kernighan BW. An e"ec-tive heuristic algorithm for the traveling-salesman problem. Operations Research 21 (1973), p. 498-516.

A BFigure 2. (A)$SDUDPHWULFFXUYHDVGUDZQRQPPJODVVFRYHUVOLGHDFFRUGLQJWRDVHWRI/LVVDMRXVHTXDWLRQV(B) 0LFURVFRSLFYLHZDVVHPEOHGIURPDPRVDLFVHWDW[PDJQLFDWLRQ

Articles


Photobiological Regulation of Chloroplast Synthesis5DFKHO6DODWNDDQG+DUYDUG/\PDQ

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-8181

Abstract

Every process that takes place in a cell is accomplished by innumerable inter-mediate steps. Chloroplast synthesis is an example of a light-mediated event. For a light-mediated process, the #rst step is the absorption of light. In Euglena gracilis, this initiates a co-translational pathway of pro-tein translocation. Both nuclear and chlo-roplast genomes are triggered to assemble chloroplasts. !e light activates a G-linked protein receptor which in turn activates the IP3 signaling pathway. IP3 signals the calcium channels in the endoplasmic reticulum (ER) to open. IP3 is then de-phosphorylated and recycled back into the membrane. !e ER releases calcium as well as inducing the transport of the chlorophyll binding proteins required for chlorophyll synthesis and chloroplast production. !e Golgi apparatus packages the protein for transport to the growing chloroplast.

Adding the inhibitor lithium chloride arrests this process by inhibiting the en-zymes which allow IP3 to be recycled back into the membrane. If the cells are washed, removing the lithium chloride, chlorophyll synthesis is resumed. Inhibiting cells al-ready in the process of creating chlorophyll binding protein causes an accumulation of these proteins inside the ER. A protein block will lead to the ER stress response being activated. Washing the cells at vari-ous times during inhibition removes the in-hibitor, allowing the cells to continue chlo-rophyll synthesis. !ere is a pronounced lag in the resumption of chlorophyll synthesis. Keeping the inhibitor in the cells for lon-ger periods of time causes increasing lags in resumed chlorophyll production. !ese lags probably correlate with the steps of the ER stress response.

Introduction

Communication within cells is orches-trated using second messenger systems. In a

light-mediated system, such as chlorophyll synthesis in Euglena gracilis, the #rst signal is activation by light. !e light initiates a chain reaction eventually leading to photo-synthesis inside the chloroplast. However, in the dark Euglena do not develop their chloroplasts beyond pro-plastids [1]. In the presence of light, the pro-plastid increases its internal membrane surface area, which folds inwards to become thylakoids. !e chloroplast and nuclear genomes begin transcribing chlorophyll-binding proteins as well as hundreds of other nuclear-coded chloroplast proteins. !e nuclear-coded proteins will need to be transported to the growing chloroplast before it can be com-pleted. !erefore, light initially signals the cell to complete the synthesis of the chlo-roplast, a process that will continue until biosynthesis is completed.

As the chloroplasts #nish their de-velopment in the light, they require the chlorophyll-binding proteins for comple-

tion. Chlorophyll itself is synthesized in the chloroplast but requires the chlorophyll-binding proteins to be inserted into the chloroplast membranes [2]. !ese proteins are coded in the nuclear genome and, af-ter transcription, are transported co-trans-lationally to the growing chloroplast [2]. For this reason, the proteins are translated on ribosomes sitting on the endoplasmic reticulum (ER) and then transported to the Golgi apparatus where they are pack-aged and sent to the chloroplast [2] (Fig-ure 1). As the proteins are elongated into the ER, the nascent chains are inhibited from further elongation by calreticulin, a calcium binding complex [3,4]. !e release of the calreticulin from the nascent chain is brought about by another simultaneous biochemical pathway initiated by light.

In Euglena, the light receptor for chlo-roplast synthesis, as well as the events it activates, lies on the outer membrane of the growing chloroplast. !e receptor di-rectly activates a G-protein linked receptor, which in turn activates phospholipase C [5]. !is enzyme cleaves phosphotidylino-sitol (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) [5]. IP3 moves away from the membrane and binds to a receptor on the ER, signaling the cal-cium channels to open. After signaling, the IP3 has its phosphate groups removed by

Figure 1. 7KHSDWKZD\RIELRV\QWKHVLVIRUFKORURSK\OOELQGLQJSURWHLQVWDUWLQJZLWKDFWLYDWLRQRIWKHOLJKWUHFHSWRURQWKHFKORURSODVWPHPEUDQHDQGWKHWUDQVFULSWLRQRIFKORURSK\OOELQGLQJSURWHLQP51$LQWKHQXFOHXV2QWKH(5ERXQGULERVRPHLVVKRZQWKHLQKLELWLRQRISURWHLQV\QWKHVLVGXHWRFDOUHWLFXOLQDQGFDOFLXP

9

Articles


three di"erent enzymes before being re-cycled back into the membrane [5].

In the ER, calcium begins to leave through the channels. As the calcium leaves the ER, the repression caused by calcium-binding calreticulin is released. Previously repressed chlorophyll-binding proteins are sent to the Golgi where they are packaged and sent out in vesicles [6]. Upon arrival at the chloroplasts, the calcium, liberated from the ER, is required for vesicle-vesicle fusion with the outermost membrane. !e greening process begins when the chlo-rophyll-binding protein successfully binds chlorophyll.

Inhibition of any step in the process stops chlorophyll synthesis. !is can be done by the addition of lithium chloride to Euglena cells. Lithium is a competitive inhibitor of the enzymes which cleave the phosphates (Ki of binding is 1 mM) [5]. !erefore, the inositol cannot be recycled into the membrane to reform IP3 (Figure 2). When the cell has run out of signaling IP3 (roughly 1% of IP3 in membranes) the cell cannot propagate the signal any further [5]. If the cells are washed in fresh medium, removing the lithium chloride, chlorophyll synthesis can be resumed.

!e washing method was originally used as proof that the cells stopped pro-ducing chlorophyll as a result of inhibition, not death. However, it was noted that after being washed the cells have a lag period

before they begin to produce chlorophyll again. !e reason for this lag was hypoth-esized to be due to the ER quality control mechanism. Normally the ER stress re-sponse would be invoked because of exces-sive amounts of mis-folded proteins in the ER [7]. !e ER cannot function properly with so many proteins clogging it. In this situation, the ER #rst sends a signal to up regulate transcription of chaperone pro-teins to assist in folding the new proteins [8]. However, if the proteins cannot be correctly folded, the additional chaperone proteins exacerbate the aggregation in the ER. In this case, the chaperone proteins work as a signal to have the excess proteins degraded [8]. A speci#c protein called BiP responds to the excessive number of un-folded proteins [7]. Under normal condi-tions, BiP binds to PERK keeping it in an inactive state [8]. When BiP leaves PERK to assist in protein folding, PERK becomes active [8]. In its active state, PERK sends a signal to the nucleus to down-regulate general protein translation [8]; reducing the amount of proteins coming into the ER gives the ER space to degrade or export its protein load.

!e chlorophyll-binding proteins are not mis-folding in the ER, but there is evidence that they build up [1]. Although the lithium chloride stops the ER channels from being opened, chlorophyll-binding proteins are still being transcribed. !ere-

fore, the nascent chains accumulate in the ER where calcium-bound calreticulin in-hibits their translation. It is logical that the ER would initiate the stress response to regain equilibrium [9]. To test this hypoth-esis, the cells were washed at three di"erent times after lithium chloride had been add-ed. !e expectation was that cells incubated in lithium chloride for longer amounts of time would have a longer lag period. !e ER in these cells would have engaged the quality control mechanism for a longer time resulting in fewer proteins stored in the ER.

Methods and Materials Euglena gracilis was grown at 31oC

in enriched, sterile medium under 900 lux (illuminance). Stocks were grown in the dark. After 5 days, both the control and experimental stocks were moved into the light. !is was marked as time zero. At 28 hours, 0.28 millimoles of lithium chloride was added to the experimental $ask. At 31, 34, or 46 hours, the lithium chloride was washed from the cells using sterile medi-um and the washed cells were placed in a sterile $ask with 10 mL of fresh medium. !e experiment was run for 96 hours. !e amount of chlorophyll was measured by centrifuging one mL of cells and pour-ing o" the supernatant. !e pellet of cells was mixed with one mL of acetone and centrifuged again. !e absorbency of the supernatant was recorded at 480, 645, 652, and 663 nanometers. Chlorophyll concen-tration was determined by the method of Arnon [1]. In a steady-state experiment, lithium chloride was added at 72 hours and the cells were not washed. !e experiment ran to 130 hours.

5HVXOWV

When $asks are placed in the light, experimental and control $asks accumulate chlorophyll at the same rate until lithium chloride is added to the experimental $ask (Figure3).At this time, chlorophyll pro-duction in the lithium $ask levels o" and eventually declines. When lithium chloride is washed from the cells there is a lag be-fore chlorophyll production begins again. As the amount of time between lithium ad-dition and washing increases, the lag time increases as well (Figure 4). In another ex-

Figure 2.,3LVUHF\FOHGEDFNLQWRWKHPHPEUDQHDIWHUVLJQDOLQJWKH(5/LWKLXPLQKLELWVWKHSKRVSKDWHJURXSVIURPEHLQJUHPRYHGWKXVLQKLELWLQJUHF\FOLQJRI,3LQWRWKHPHPEUDQH

Articles


periment, both the control and experimen-tal $ask were allowed to green to a state when chlorophyll is made at a relatively constant rate. When lithium was added at this time the cells showed the same de-clining response in chlorophyll production (Figure 5).

Discussion

!e decreased production of chloro-phyll in the lithium-inhibited cells is de-pendent on the binding a%nity of lithium chloride to the three phosphatases that

cleave IP3. Since not every enzyme is bound to a molecule of lithium at every moment, small amounts of chlorophyll are still made. Increasing the concentration of lithium would decrease the amount of chlorophyll produced.

!e lithium-inhibited cells do not im-mediately drop in chlorophyll synthesis because at the onset of lithium addition some IP3 is attached to the pro-plastid membrane. !e remaining signaling IP3 leaves this membrane and opens the calci-um channel even in the presence of lithium. !erefore, chlorophyll levels continue to

increase until the cells runs out of signaling IP3. !e chlorophyll level plateaus when the protein product is being made but no new signal is created. Chlorophyll levels only begin to drop when no more new pro-tein can be made.

!e lag between when chlorophyll amounts plateau and when chlorophyll production decreases can be attributed to the ER stress response. !e increasing lag times between 3, 6 and 18 hour washes may be due to the stages of the response. When the cells were washed 3 hours after lithium was added the ER had sent a signal to the nucleus to down-regulate transcription of chlorophyll binding protein as well as up-regulate chaperone proteins [8]. !erefore, when the lithium chloride was washed out the cell had only to transport the chloro-phyll binding proteins already in the ER to the Golgi and then the chloroplast. !e cells washed at 6 hours appear to be a simi-lar situation within the ER. However, the cells washed at 18 hours have a consider-ably longer lag than the other two. !is would be due to the degradation of proteins in an e"ort to promote the ER response. When the cells were washed there was a limited amount of chlorophyll binding pro-tein already in the ER. Transcription had to be up-regulated in the nucleus and new proteins made from mRNA before new chlorophyll was seen in the cells.

Another consideration for the lag in chlorophyll production is fate of the sig-naling IP3. Highly active IP3 sitting in the cytoplasm will be degraded before the cells are washed. After lithium is removed, the cells must synthesize more signaling IP3. It should also be noted that after activation the IP3 receptor on the ER is often marked for degradation [10]. It is possible for IP3 to be active in the cell without an available ER receptor due to degradation. !e cycle of this system could also a"ect the kinetics of chlorophyll-binding protein synthesis.

!e #nal experiment in which lithi-um chloride was added after the cells had reached a steady state of chlorophyll syn-thesis displays the dynamic equilibrium of chloroplast production. Although the level of chlorophyll remains more constant at this point, adding inhibitor still results in decreased chlorophyll production. Chlo-rophyll has a limited lifetime, after which the function is compromised and the chlo-rophyll is degraded. A constant $ow of

Figure 3. $PRXQWRIFKORURSK\OOSURGXFHGLQFHOOVDIWHUEHLQJSODFHGLQWKHOLJKW7LPHRIOLWKLXPDGGLWLRQPDUNHGE\&HOOVZHUHZDVKHGDWDQGKRXUVDIWHUOLWKLXPZDVDGGHGDVQRWHGLQFKDUWWLWOH

11

Articles


chlorophyll binding protein is therefore required as long as the cell is undergoing photosynthesis. !e increased lag times are not due to a natural decline in chlorophyll biosynthesis but to the active down-regula-tion and degradation of the protein.

Conclusion

!e addition of lithium chloride to Euglena gracilis successfully inhibits chlo-rophyll synthesis, while washing cells re-verses the inhibition. !e lag in chlorophyll synthesis after the inhibitor is removed is lengthened based on the amount of time chloroplast synthesis is inhibited. !e lags seen probably constitute steps in the endo-plasmic reticulum stress response.

5HIHUHQFHV

1.Lyman, Harvard, Masahiro Mitsuboshi, Asami Endo, Kazuyuki Tanaka, and Tet-suaki Osafune. !e Regulation of Chloro-plast Synthesis by a Light Mediated Phos-phoinositide System. Bull. of Nippon Sport Sci. Univ. 30 (2000): 141-52.2.Sulli, Chidananda, ZhiWei Fang, Umesh Muchhal, Steven D Swartchbach. Topol-ogy of Euglena Chloroplast Protein Pre-cursors within Endoplasmic Reticulum to Golgi to Chloroplast Transport Vesicles. !e Journal of Biological Chemistry 274 (1998): 457-463.3.Jia, Xiao-Yun, Li-Heng He, Rui-Lian Jing, Run-Zhi Li. Calreticulin: conserved protein and diverse functions in plants. Physiologia Plantarum 136 (2009):127-

138.4.Navazio, Lorella, Maria C. Nardi, Sim-onetta Pancaldi, Paoglo Dainese, Barbara Baldan, Anne-Catherine Pitchette-Laine, Loic Faye, Flavio Meggio, William Martin, Paola Mariani. Functional Conservation of Calreticulin in Euglena gracilis. Journal of Eukaroyotic Microbiology 45 (1998): 307-313.5.Berridge, Michael J. Inositol Triphos-phate and Diaclyglycerol: Two Interacting Second Messengers. Annual Review of Biochemistry 56 (1987): 159-93.6.Van Doreen, Geil G., Steven D. Schwartzbach, Tetsuaki Osafune, Geof-frey I. McFadden. Traslocation of proteins across the multiple membranes of complex plastids. Biochimica et Biophysica Acta 1541 (2001): 34-53.7.Ellgaard, Lars, and Ari Helenius. Qual-ity Control in the Endoplasmic Reticulum. Nature Reviews 4 (2003).8.Ron, David. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 2002; 110(10):13839.Ma, Yanjun, and Linda Hendershot. ER Chaperone Functions during normal and stress conditions. Journal of Chemical Neuroanatomy (2003).10.Pearce, Margaret M. P., Yuan Wanq, Grant G. Kelley, Richard J. H. Wojcikie-wicz. SPFH2 Mediates the Endoplasmic Reticulum-associated Degradation of Ino-sitol 1,4,5-Trisphosphate Receptors and Other Substrates in Mammalian Cells. !e Journal of Biological Chemistry 28 (2007): 20104-20115

Figure 4. 7LPHODSVHEHWZHHQZKHQOLWKLXPZDVZDVKHGIURPFHOOVDQGZKHQQHZFKORURSK\OOZDVSURGXFHGDVEDVHGRQ)LJXUH

Figure 5. &HOOVZHUHOHIWLQWKHOLJKWXQWLOFKORURSK\OOSURGXFWLRQZDVOHYHO/LWKLXPZDVDGGHGWRH[SHULPHQWDODVNDURXQGKRXUVPDUNHGE\

Reviews


(in children) [3]. Untreated or inadequately treated Lyme disease can lead to severe neurological, cardiac, and/or arthritic compli-cations. Bells palsy, which is paralysis of the facial nerve, is an example of a neurological manifestation of borreliosis. Lyme dis-ease, however, has been seen to manifest itself di"erently around the world - in Europe, patients most frequently exhibit neurologi-cal symptoms, or neuroborreliosis, whereas in North America ar-thritis prevails [2]. Furthermore, it should be kept in mind that Lyme borreliosis is only one disease out of many that are caused by other tick-borne viruses and bacteria, such as spotted fever rickett-siae, babesia, ehrlichia, bartonella, anaplasma and mycoplasma [6]. People displaying persistent symptoms of Lyme disease are often found to have co-infections of one or more of these other bacte-ria. Diagnosis is based on clinical symptoms and laboratory tests, such as Western blots and Enzyme-linked immunosorbant assays (ELISA), although reaching a de#nite diagnosis is often extremely complicated [3]. In addition, the laboratory tests have been de-scribed by some physicians as unreliable, and a seronegative patient may still be su"ering from Lyme disease [3,7].

!e B. burgdorferi spirochete contains over 1,500 genes and 21 plasmids, the highest number found in any known bacterium, which allow B. burgdorferi to adapt rapidly to changes in the envi-ronment [8]. It can penetrate into the mammalian central nervous system and invade cells such as #broblasts, synovial cells, endothe-lial cells, and macrophages.

In addition, the bacteria can become resistant to treatment by assuming a non-replicating cyst-form, which can become ac-tive months after the initial infection [8]. Traces of the bacteria have been found in the ligaments of patients who had been su"er-ing from Lyme disease for years [9]. PCR results have con#rmed the presence of B. burgdorferi DNA in cerebrospinal $uid, urine, blood, skin, and synovial $uid and tissue as well as in the muscles of patients with persistent Lyme disease [10]. In order to type the bacteria, two markers have been used by researchers - OspC, an outer surface protein, and ribosomal RNA spacer typing (RST) that #nds a unique region in RNA speci#c to the bacteria. OspC

Lyme Disease: An Overview1DG\D3HUHVOHQL

!e words Lyme disease bring to mind pictures of the red rings of a bulls eye rash - a classic sign of early infection. However, there is much more to this disease than meets the eye. First iden-ti#ed in 1975 after an outbreak of juvenile rheumatoid arthritis in the town of Lyme, Connecticut, Lyme disease has been slowly revealed to be an extremely complicated and treacherous infection. In fact, it is now known to be the most common tick-borne disease in the Northern Hemisphere [1]. In 1982, Dr. Willy Burgdor-fer discovered the culprit from studying deer ticks - a bacterium, named B. burgdorferi in honor of the discoverer. B. burgdorferi is a spirochete, a spiral-shaped bacterium, that lives in the gut of Ixo-dus ticks and is successfully transmitted to humans by both nymph and adult ticks about 48 hours after the bite [2,3]. !ere are mul-tiple species of Borrelia bacteria - in North America, for example, Lyme disease is caused by B. burgdorferi, but in Europe it can also be caused by its cousins, B. afzelii and B. garinii [4]. In the United States, endemic areas include Connecticut, Delaware, Maryland, Massachusetts (especially Cape Cod), Minnesota, New Jersey, New York (especially Long Island), Pennsylvania, Rhode Island and Wisconsin [5]. Ticks are most common at ground level, in grass and low shrubbery. It should be a habit, therefore, for hik-ers and campers to wear light colored, long-sleeved clothing and check themselves for ticks at the end of the day.

!e major clinical symptoms of Lyme borreliosis are numer-ous, often worsening in cyclical intervals. !ey include $u-like symptoms, such as muscle and joint pain, fever, fatigue, nausea and #bromyalgia, along with more serious neurological and psychiatric symptoms such as memory loss, myocarditis, palpitations, head-aches and neck sti"ness. !e bulls eye rash - erythema migrans - occurs in about 50% of patients. !e B. burgdorferi infection can be successfully cured in the majority of cases with promptly administered antibiotics such as doxycycline, one of the macrolides (erythromycin and its derivatives), ceftriaxone, and/or amoxicillin

Figure 1.,[RGXVWLFNVWKDWFDUU\%EXUJGRUIHUL$QWLPLFURELDO$JHQWV&KHPRWKHUDS\

KWWSLQVWUXFWZHVWYDOOH\HGXJUDQLHULWLWHOBELJMSJ

Figure 2.B.burgdorferi VSLURFKHWHV

13

Reviews


system damage [20].A great deal of research has been done at di"erent universities

on the mechanisms of borreliosis and the resulting cellular in$am-matory response. At Stony Brooks Center for Infectious Diseases, Borrelia burgorferi has been studied for almost two decades in the laboratory of Dr. Jorge Benach. Dr. Benach, chair of the Depart-ment of Molecular Genetics and Microbiology, has been studying the mechanisms of antibody function on B. burgdorferi. !e group discovered that one of the bacterias outer-surface proteins, OspB, is recognized by one of the bodys bactericidal antibodies, CB2, which then lyses the bacteria through pore formation [21]. Dr. Benachs research has elucidated the mechanism by which CB2 recognizes OspB through a Lys-residue on the carboxy terminus of the protein. !is is a unique and important result because very little is known about the mechanism of bactericidal antibodies, and their defensive role may carry potential for developing future treat-ments.

Along with Dr. Benachs work, the laboratory of Professor Martha B. Furie has published on the role of interferon-gamma (IFN-gamma), a cell-signaling molecule of the immune system, in the endothelial tissue of a genetically engineered mouse model that was infected with B. burgdorferi. IFN-gamma is like a molecular switch that turns on chronic in$ammation, explained Dr. Furie. When the bacteria disseminate throughout the body after the tick bite, they activate the endothelium and begin the in$ammatory process by attracting T lymphocytes that secrete IFN-gamma [22]. !e in$ammation was found to be due, in large part, to a synergis-tic e"ect of B. burgdorferi and IFN-gamma, which together acti-vate the transcription of a series of genes in endothelial cells. !ese genes encode chemokines, or chemoattractants, speci#c for T lym-phocytes. Interestingly, there seemed to be selection for those T lymphocytes that secreted more IFN-gamma, and the result was a positive feedback loop that generated more and more IFN-gam-ma, leading to a state of chronic in$ammation in the tissue. !e damage to human tissues is likely caused by the bodys reaction to the bacteria, not the bacteria themselves [22].

Today, immunologic studies have joined forces with neurolog-ic and clinical assessments of both groups of infected patients and animal models to study the twists and turns of B. burgdorferi. !e spirochete remains both a serious infectious threat to the public and a tantalizing puzzle to scientists. !e development of a suc-cessful vaccine would be an immense accomplishment. So far, a failed vaccine called LYMErix was removed from the market in 2002 due to patient concern over side e"ects [23]. Various re-search has been done on other options - just recently, scientists at Yale University showed that antiserum to Salp15, a protein found in tick saliva that shields the tick from mammalian immune re-sponse, successfully protected mice from B. burgdorferi infection [24]. !us, there is hope that with such promising research and continued collaboration within the scienti#c community, a much needed solution to Lyme disease will be developed in the not so distant future.

typing divides the bacteria into 21 types, while RST divides them into just 3 groups. !e RST1 strain was, in addition, found most frequently in joint $uid of patients who did not respond to antibi-otic treatment [11].

Over the past years, there has been debate in the scienti#c and medical #elds on proper treatment of the disease. !e disagree-ment centers on the usefulness of prolonged antibiotic treatment for patients whose symptoms, such as fatigue, cognitive impair-ment, and arthritis, do not go away after 14-21 days of treatment. Persistent symptoms were found in 53% of cases in a study that was done with serologically positive patients who were evaluated three years after infection [12]. However, there have been very few antibiotic therapy trials, and the ones that do exist have shown contradicting results. For example, Fallon et al. showed that yes, prolonged antibiotic treatment results in better cognitive scores unless it is discontinued [13,14], whereas two other studies showed improvement in fatigue, but not cognition [15,16]. Each of the clinical trials, however, was performed under di"erent parameters and none of the trials noted the duration of infection and symp-toms in the participants [17]. !is problem highlights the need for more controlled clinical trials to be conducted before a #nal deci-sion is reached on the usefulness of long-term treatment.

Along with clinical assessments, over twenty years of research have begun to resolve the multiple perplexities of B. burgdor-feri. Published results have shown that Borrelia spirochetes can be found in patients even after treatment, with evidence that the bacteria can change into cyst forms that are not a"ected by certain types of antibiotics [18]. Experiments on adult Macaca mulatta primates revealed that B. burgdorferi in the central nervous system localized to nerve roots, leptomeninges (the innermost layers that surround the brain) and dorsal root ganglia, which relay sensory information into the spinal cord from the rest of the body [19]. An additional primate model that evaluated B. burgdorferi-infected rhesus monkeys six months after infection revealed the presence of Borrelia antigens identi#ed by Western blot analysis and PCR, with observed arthritis of the knee and elbow joints, joint structur-al changes, demyelinization and other signs of peripheral nervous

Figure 3.B. burgdorferiGHYHORSVJUDQXOHVF\VWVZLWKHQYLURQPHQWDOVWUHVV

$QWLPLFURELDO$JHQWV&KHPRWKHUDS\

Reviews


5HIHUHQFHV1. Vojdani A, Hebroni F, Raphael Y, et al. Novel Diagnosis of Lyme Disease: Potential for CAM Intervention. 2009. Evid Based Complement Alternat Med. 6(3):283-95. 2. Rosner, Bryan. !e Top 10 Lyme Disease Treatments. 2007. BioMed Publishing Group. p316.3. Donta ST, MD. Late and Chronic Lyme Disease. 2002. Medical Clinics of North America. Vol 86:(2):341-9, vii.4. Steere AC, Gross D, Meyer A et al. Autoimmune Mechanisms in Antibiotic Treatment-Resistant Lyme Arthritis. 2001. J Auto-imm. 16, 263-268. 5. Map: National Lyme disease risk map. Apr 28, 2004. www.cdc.gov.6. Hamlen, R. Lyme borreliosis: perspective of a scientist-patient. 2004. !e Lancet: Infectious Diseases. Oct, Vol. 4: 10 (603-604).7. Dattwyler RJ, Volkman DJ, Luft BJ et al. Seronegative Lyme disease. Dissociation of speci#c T- and B- lymphocyte respons-es to Borrelia burgdorferi. 1988. N Engl J Med. 319:1441-6.8. Phillips SE, Harris NS, Horowitz Ret al. Lyme disease: scratch-ing the surface. 2005. !e Lancet. Vol 366.9. Hupl T, Hahn G, Rittig M et al. Persistence of borrelia burg-dorferi in ligamentous tissue from a patient with chronic lyme bor-reliosis. 2005. Arthritis & Rheumatism. 36(11); 1621-1626.10. Frey M, Sibilia J, Piemont Y et al. Detection of B.burgdorferi DNA in Muscle of Patients with Chronic Myalgia Related to Lyme Disease. Amer J Med. 104(6);591-594.11. Fiore, K. Strain Di"erences Associated with Refractory Lyme Arthritis (Review of Allen C. Steeres article in July on Arthritis and Rheumatism). 2009. Medpage Today.12. Asch ES, Bujak DI, Weiss M et al. 1994. Lyme disease: an in-fectious and postinfectious syndrome. J Rheumatol; 21(3):454-61.13. Fallon BA, Keilp JG, Corbera KM et al. A randomized, pla-cebo-controlled trial of repeated IV antibiotic therapy for Lyme encephalopathy. 2008. Neurology. 25;70(13):992-1003. 14. Cameron, D. Severity of Lyme disease with persistent symp-toms. Insights from a double-blind placebo-controlled clinical trial. 2008. Minerva Med; 99(5):489-96.15. Krupp LB, Hyman LG, Grimson R et al. Study and treatment of post Lyme disease (STOP-LD): a randomized double masked clinical trial. 2003. Neurology;60:1923-30.16. Kaplan RF, Trevino RP, Johnson GP, et al. Cognitive function in post-treatment Lyme disease: do additional antibiotics help? 2003. Neurology;60:1916-22. 17. Cameron DJ. Generalizability in two clinical trials of Lyme disease. 2006. Epidemiol Perspect Innov. 17;3:12.18. Mursic VP, Wanner G, Reinhardt S et al. Formation and cul-tivation of Borrelia burgdorferi spheroplast-L-form variants. Infec-tion. 1996 May-Jun;24(3):218-26.19. Cadavid D, ONeill T, Schaefer H, Pachner AR. Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease. 2000. Lab Invest;80(7):1043-54.20. Roberts ED, Bohm RP Jr, Lowrie RC Jr. et al. Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early dis-seminated and chronic phases of disease in the peripheral nervous system.1998. J Infect Dis;178(3):722-32.

21. Anderton JM, Tokarz R, !ill CD et al. Whole Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody. 2004. Infect and Immunity. 72(4): 2035-2044.22. Dame TM, Orenzo" BL, Palmer LE, Furie MB. IFN-y Al-ters the Response of Borrelia burgdorferi-Activated Endothelium to Favor Chronic In$ammation. 2007. J Immun. 178:1172-1179.23. Abbott A. Lyme disease: Uphill Struggle. 2006. Nature 439, 524-525.| doi:10.1038/439524a24. Dai J, Wang P, Adusumilli S, Booth CJ et al. Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent. 2009. Cell Host Microbe. 6(5):482-92.

15

Reviews


Cocaine Addiction: Understanding its Pharmacological Basis and Psychological Effects$OOLVRQ*ROGEHUJ

How Cocaine Works

Dopamine (DA) is a neurotransmitter within the Central Nervous System which plays many roles. Its transmission can affect behavior and cognition. Voluntary movement, mood, sleep, working memory, and punishment and reward learning are just some of the processes that are regulated by dopami-nergic transmission [1].

Dopamine is released by pre-synaptic neurons into the synaptic cleft and then bind to dopamine receptors on post-synaptic neurons [2]. The performance of tasks associated with dopaminergic transmission rely on proper maintenance of dopamine levels present in the synaptic cleft. Cocaine dis-rupts this maintenance of DA levels in the synaptic cleft by inhibiting dopamine transporters (DAT), which normally function as dopamine re-uptake transporters. DAT moves DA from the synaptic cleft back into the pre-synaptic neu-ron where it can no longer interact with post-synaptic recep-tors and elicit responses associated with dopaminergic signal transmission. Cocaine binds to the transporter DAT1 and the complex can no longer function to uptake DA. The high associated with taking cocaine is a result of this major in-crease in dopaminergic transmission due increased levels of dopamine in the synaptic cleft interacting with post-synaptic receptors [3].

The Development of Tolerance and Addiction

Tolerance is a common issue that evolves with continued use of a drug. Habitual cocaine use results in recurrent situ-ations of excess dopamine in the synaptic cleft, which then bind to post-synaptic receptors. In response to this continual excess of dopamine in the synaptic cleft, a down-regulation of dopamine receptors occurs. This leads to fewer dopamine D2 receptors on post-synaptic neurons. As a result, it is more difficult to achieve the same level of dopaminergic transmis-sion originally experienced with cocaine use. Additionally, this leads to disruption of normal dopaminergic transmission (without cocaine), as the amount of DA maintained in the synaptic cleft is no longer adequate to elicit the same trans-mission that occurred when a greater number of receptors were present [4].

In addition to the down-regulation of dopamine D2 re-

ceptors, a reduction in DA release and transmission, as a re-sult of natural stimuli, has been seen in drug abusers [5]. This lessening of natural dopamine transmission and decreased number of D2 receptors contribute to the desire for users to re-administer cocaine after it has worn off, as without the drug, natural stimuli cannot elicit the same level of transmis-sion as they do in a non drug addicted individual [1]. This decrease in dopaminergic transmission due to natural stimuli is a major factor in why addicts have such trouble quitting.

The Neurobiology Behind Cocaine Addiction

A group of researchers at Brookhaven National Labora-tory (BNL) have been focusing on the neurobiological basis for the psychological symptoms of cocaine addicted indi-viduals. It has previously been found that the orbitofrontal cortex (OFC) and cingulated gyrus (CG) are involved in the regulation of motivation and drive. Enhanced activation of the OFC and CG occurs with drug-induced DA stimulation. This enhanced stimulation of regions known to regulate mo-tivation and drive could lead to an increased drive for cocaine addicts to self-administer the drug [6]. Additionally, the OFC has been found to be involved with stimulus-reward reinforcement learning. Its hyper-activation in drug addicted subjects suggests it is a neural pathway of reward predic-tion, which contributes to drug cravings in drug addicted individuals [1]. In addition to the development of cravings, cocaine addicted individuals have been found to give dis-proportionate value to their drug while disregarding other stimuli which are seen as rewarding by non-drug addicted individuals. This was seen in a study which found more than half of cocaine abusers rated a $10 reward equally valuable to a $1000 reward [7]. This assignment of greater value to the drug than to non-drug stimuli and lack of ability to recog-nize a gradient of reward among non-drug stimuli may play a role in leading cocaine addicts to make decisions which are recognized as disadvantageous by normal individuals. An example given by Dr. Goldstein at BNL: trading a car for cocaine [8].

&RQWLQXHG5HVHDUFK

Drug addicts have always displayed abnormal behaviors, but it is only with current research on the neurobiology be-hind the psychological changes in addicts that we are be-ginning to understand the basis of their condition and the importance of drug addiction as a scientific issue and not just a societal disturbance. Based on the behavioral and fMRI studies conducted in Dr. Goldsteins laboratory at BNL and in laboratories at Stony Brook University and the National Institute on Drug Abuse, it is becoming clear that neuro-logical pathways in cocaine addicted individuals become al-tered from that of normal individuals [5]. These alterations in neurotransmission contribute to addiction and the psy-chological symptoms seen in drug addicted individuals. It is this finding which serves as the basis for continued research into the neurobiology of the development of addiction and

Reviews


the neuronal alterations caused by drug addiction, which will lead researchers to better treatments for drug addiction.

5HIHUHQFHV

1. Volkow ND, Fowler JS, Wang GJ, Goldstein RZ (2002) Role of dopamine, the frontal cortex and memory circuits in drug addiction: insight from imaging studies. Neurobiol Learn Mem 78: 610624.

2. Landes, NM. How Does Cocaine Alter Neurotrans-mission. National Institutes of Health, National Institute on Drug Abuse. Bethesda, MD. 2000. Video.

.

3. Berrios, Miguel. Adrenergic Drugs. BCP 401: Prin-ciples of Pharmacology. Department of Pharmacology, Stony Brook University Medical Center, Stony Brook, NY. 11 Nov. 2009. Lecture.

4. Berrios, Miguel. CNS Pharmacology. BCP 401: Principles of Pharmacology. Department of Pharmacology, Stony Brook University Medical Center, Stony Brook, NY. 9 Nov. 2009. Lecture.

5. Goldstein RZ, Volkow ND (2002). Drug addiction and its underlying neurobiological basis: Neuroimaging evi-dence for the involvement of the frontal cortex. Am J Psy-chiatry 159: 16421652.

6. Tucker, DM, Luu, P, & Pribram, KH. (1995). So-cial and emotional self-regulation. Annals of the New York Academy of Sciences, 769, 213239.

7. Goldstein RZ, Tomasi D, Alia-Klein N, Cottone LA, Zhang L, Telang F et al (2007b). Subjective sensitivity to monetary gradients is associated with frontolimbic activa-tion to reward in cocaine abusers. Drug Alcohol Depend 87: 233240.

8. Brookhaven National Laboratory. Medical Research Department. Altered Perception of Reward in Human Co-caine Addiction. Laboratory News. Department of Energy, 15 Oct. 2006. Web. 12 Mar. 2010. .

17

Reviews


Stem Cells: A Therapeutic Hope

Reviews


the Notch pathway in elderly mouse because the muscle cells of each respective mouse did not crossover [11, 12]. Current research is attempting to utilize ES cells to reactivate the Notch pathway in older muscle in order to promote regeneration [13].

Blood

Hematopoietic stem cells, which are found in the bone mar-row, gives rise to blood cells, which are vital for oxygen transport throughout the body via hemoglobin (red blood cells) and play a role in the immune response (white blood cells). !ey also possess a high turnover rate and regenerative capacity with life spans of 10 days [11]. Studies have shown that a single blood stem cell can produce all of the blood in an animal [14]. !is was shown in an experiment performed by Drize et al., where the blood supply in a mouse was #rst irradiated. !en when a single blood stem cell was injected, the mouse survived because all of the blood cells reformed from that one cell. Currently, adult stem cells are used in a clinical capacity, speci#cally in bone marrow transplants [3]. In February 2010, scientists at UC San Diego identi#ed the speci#c vertebrate region where adult stem cells arise during embryonic development. !is provides insight into the di"erentiation pathway between ES cells and adult stem cells, and could lead to future therapies for pa-tients su"ering from blood disorders by producing patient-speci#c hematopoietic stem cells [15].

Recently, scientists in Weill Cornell Medical School discov-ered a new method of propagating blood stem cells, which en-

hanced the life of these cells by 21 days or more. Endothelial cells were used in this study because they create a microenvironment that triggers the regeneration of blood stem cells in the bone mar-row [16]. For this study, the gene, E4ORF1, from an adenovirus was inserted into endothelial cells. !ese genetically-modi#ed cells were then propagated in a petri-dish with blood stem cells extract-ed from mice without the addition of serum or growth factors. !is resulted in the production of a large number of hematopoietic stem cells. When these cells were inserted into mice with irradi-ated blood cells, these cells di"erentiated into all of the necessary blood cells within the mouse. !ere were no signs of tumor devel-opment within these animals after more than a year. !is method was highly e"ective because by eliminating the growth factors, the stem cells were only in$uenced by the signals from the endothelial cells, which dictated di"erentiation into blood cells. As a result, this decreases the possibility of tumor formation or improper cel-lular di"erentiation. Scientists are hoping to use this knowledge to devise therapies of inhibiting tumor proliferation in cancer pa-tients [16].

Diabetes

Diabetes mellitus is an autoimmune disorder where the beta cells of the pancreas are destroyed and thus fail to produce insulin (a vital hormone that signals cells to uptake glucose), resulting in high glucose level build-up in the bloodstream. Since the pancreas possesses a low cellular turnover and high regenerative potential

KWWSZZZEXPFEXHGXVWHPFHOOVOHVSLFWXUHMSJIn vitroFXOWXUHRIHPEU\RQLFVWHPFHOOVGHULYHGIURPPLFH

19

Reviews


[11], it has a limited regenerative capacity. In 2000, scientists in University of Wisconsin in Madison

showed that ES cells can di"erentiate and express the insulin gene in human patients [17]. In 2001, scientists di"erentiated mouse embryonic cells into structures that resembled pancreatic islets that are able to secrete beta cells [18]. Current research is focused on the introduction of ES cells into the pancreas in hopes of promot-ing their di"erentiation into beta cells to regain pancreatic insulin secretion capability [19].

Recently, Professor Anthony Atala of Wakeforest University School of Medicine has discovered amniotic $uid cells, which are in a state intermediate of embryonic stem cells and adult stem cells and possess the potential to di"erentiate into any cell in the hu-man body [20]. Although the therapeutic potential of these cells is currently being evaluated, studies have shown that these cells do not form teratomas [21]. Atalas team is currently attempting to coax these cells to di"erentiate into beta cells of the pancreas [20].

&DUGLRORJ\2UJDQ5HJHQHUDWLRQ

Although it was believed that the heart possesses a low re-generative capacity due to its low cellular turnover rate and regen-erative potential [11], recent studies have indicated that the heart possesses a minute reservoir of progenitor cells that are capable of some regenerative capacity [22], when Antonio Beltrami #rst iso-lated c-kit+ cells from the myocardium of an adult rat [23]. !ese cells have the capacity to self-renew, are both multipotent and clo-nogenic, and give rise to three di"erent cardiogenic cell pheno-types (cardiomyocytes, smooth muscle cells, and endothelial cells). When these cells were injected into rat hearts after myocardial in-farction, the myocardium was regenerated [24].

Heart formation results when pluripotent cells di"erentiate into mesodermal progenitors, where pluripotent capability de-creases and di"erentiation capacity increases due to increased ac-tivity of lineage-speci#c activators, such as brachyury and MESP from the microenvironment. From this stage, cardiac cell fates are determined when they are separated into distinct popula-tions of cardiac progenitor cells, distinguished by the presence of NKX2.5 alone (#rst heart #eld progenitors) or a combined expres-sion of NKX2.5 and ISL1 (second heart #eld progenitors). !e fates of these progenitors are determined by additional transcrip-tion factors. First heart #eld progenitors di"erentiate into car-diac conduction cells (HF-1b expression) and cardiac muscle cells (GATA4 expression), while second heart #eld progenitors di"er-entiate into cardiac muscle cells (GATA4 expression), endothelial cells (HOXB5 expression), and postnatal cardiac progenitors [22]. When scientists attempted to increase the number of progenitor cells through the injection of ES cells, teratomas developed due to high expression of a multitude of growth factors from the cardiac microenvironment, causing the ES cells to assume a multitude of cardiac fates simultaneously [24]. To avoid such malignant results, scientists are attempting to coax ES cells into assuming a particular cardiac di"erentiative fate prior to injection by exploiting cellular pathways, such as the IGF-1/PI3 kinase/Akt signaling pathway, to control their proliferation [24]. In addition, the use of ES cells has been shown to provoke an immune response, hindering the e"ec-tiveness of the treatment [25]. When Cohen and Leor used a scaf-

fold to transfer ES cells into the scar tissue of a rat after myocardial infarction, they observed enhanced cardiac repair as compared to regular injection [26] because the sca"old was site-speci#c and it gradually transferred ES cells into the site of injury, reducing the risk of teratomas.

Last year, scientists at the Mayo Clinic were the #rst to use iPS cells to treat acute myocardial infarction in mice [27]. In this study, vectors with the four human transcription factors were packaged into a plasmid and transduced into mouse embryonic #broblasts and proliferated in vitro. !ese cells were then transplanted into 8-12 week old athymic mice via four injections into the heart. !e iPS cell therapy restored myocardial performance that was initial-ly lost after the heart attack, stopped further heart damage, and regenerated damaged heart tissue at the site of injury [27]. !e success of this study shows the potential of iPS cells in cardiac repair and will allow scientists to devise patient-speci#c iPS cells for therapy in the future.

Doris Taylor has approached the question of cardiac regenera-tion from a di"erent perspective. Professor Taylors lab developed a technique called whole organ decellularization where all of the cells are stripped from an organ, leaving only the extracellular ma-trix [28]. In this technique, a heart was removed from an elderly rat, which was then drained for 12 hours with a solution to strip the heart of all of its cells. !e heart sca"old was then reseeded with the cells of the organism that would receive the transplant. Within a week, the heart was rejuvenated and began to pump be-cause the stem cells had di"erentiated into their respective roles in the organ [28].

Professor Atala employed a similar technique for organ regen-eration when he engineered a heart valve. A pig valve (since it is most similar in design to a human heart valve) was stripped of all of its cells, leaving only a sca"old behind. Next, this sca"old was placed underneath an inkjet printer that coated the valve with stem cells. !e valve was then subject to cyclic stretching, a form of exer-cise that resembles valve contractions within the body. After six weeks, the valve was ready for implantation into a human patient. !is tech-nique was employed by Professor Atalas lab in 2004, when his team developed the #rst lab-grown organ (a bladder) that was implanted into a human [29]. !us far, #ve such bladders have been transplanted into patients and all #ve work just as e"ectively as their native human counterparts [30]. !is technique is highly e"ective because it allows organs to be created that are speci#cally-suited for each patient, in terms of size and length without any fear of rejection.

In vitroFXOWXUHRILQGXFHGSOXULSRWHQWVWHPFHOOVKWWSZZZVFLHQFHSURJUHVVRUJZSFRQWHQWXSORDGVLSVBFHOOVBMSJ

Reviews


Parkinsons Disease

Parkinsons disease is the gradual loss of nigrostrial dopamine-containing neurons resulting in motor skill impairment [31]. Due the brains limited regenerative capacity [11], damage to the Cen-tral Nervous System is very di%cult to repair.

Earlier this year, scientists at Stanford University School of Medicine transformed mouse #broblasts into functioning nerve cells in vitro through the use of three transcription factors (Brn2, Myt1l, and Ascl1) via lentivirus transfection [32]. !e resulting neurons looked similar to natural neurons, were produced faster (within a week) and with an enhanced 20% e%ciency (compared to the normal 1-2% of cells that assume pluripotency in the typi-cal iPS cell protocol). !ey also skipped the pluripotent phase of development, thus preventing the formation of tumors. !ese neu-rons expressed identical function to natural neurons, including the generation of action potentials, the formation of synapses, and the expression of multiple neuron-speci#c proteins [32]. !is study suggests that the pluripotent phase maybe just a stage in embryon-ic development that could be skipped using the correct transcrip-tion factor combination [33]. !is study is a major step forward in understanding the progression of Parkinsons disease in patients and for developing patient-speci#c neurons from their own cells.

Stem Cell Institute

Since 2007, Germany has possessed a stem cell institute which o"ers stem cell treatments to patients su"ering from detrimental maladies, including diabetes, stroke, spinal injuries, arthritis, heart disease, multiple sclerosis, and many more [34]. In March of 2010, the UC Davis Center for Regenerative Cures opened in Sacra-mento, California. !is is the #rst stem cell institute in California to o"er stem cell treatments to patients. !is facility will house 200 scientists and medical personnel and will o"er treatments for heart damage, vision impairment, HIV, Huntingtons disease, and peripheral vascular disease [35]. An individualized stem cell line will be created for each patient from their own stem cells to be used for their individual therapy. !is shows just how much stem cell research has evolved since its start in 1998. It is now a sub-sector of translational medicine, where scienti#c discoveries at the bench will be applied bedside to patients in the pursuit of o"ering hope and novel treatment against todays maladies.

Conclusion

Since the late 1990s, stem cells have arisen as one of the most powerful and prominent research areas for disease therapy. Al-though the ethical debate has not been fully put to rest, the dis-covery of iPS cells opens a new door for cellular reprogramming with the quick creation of patient-speci#c stem cells for therapies. Despite the current limitations of these cells, scientists are work-ing assiduously to advance understanding and procedures to better control stem cell proliferation and di"erentiation. !e progress of this #eld in its short existence illustrates the clinical potential that these cells may have to o"er patients long-awaited therapies from their detrimental maladies. With the opening of more translational

medicine stem cell institutes and the allocation of greater funding to this #eld of research, the future of this #eld and its potential to enhance the lives of humanity looks very bright.

Acknowledgements

I thank Bin Zeng for reviewing this article and for our fruitful conversations about stem cell research.

5HIHUHQFHV

1.Smith A. A glossary for stem-cell biology. 2006. Nature. 441, 1060.2.Barrilleaux B, Phinney DG, Prockop DJ, and OConnor KC. Tissue Engineering. 2006. 12(11), 3007-3019. 3.Melton, Douglas. Understanding Embryonic Stem Cells. 2008. !e Howard Hughes Medical Institute Lecture Series..4.Kochar PG. What Are Stem Cells? 2004. ProQuest Information and Learning. .5.Bordignon C. Stem-cell therapies for blood diseases. 2006. Na-ture. 441, 1100-1102. 6.Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, Ma-ier LS, Nguemo F, Menke S, Haustein M, Hescheler J, Hasenfuss G, and Martin U. Generation of Functional Murine Cardiac My-ocytes From Induced Pluripotent Stem Cells. 2008. Circulation. 118, 507-517. 7.Okita K, Ichisaka T, and Yamanaka S. Generation of germline-competent induced pluripotent stem cells. 2007. Nature. 448, 313-317. 8.Okita K, Nakagawa M, Hyenjong H, Ichisaka T, and Yamanaka S. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. 2008. Science. 322, 949-953. 9.Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schler HR, Duan L, and Ding S. Generation of Induced Pluripotent Stem Cells Using Recombi-nant Proteins. 2009. Cell Stem Cell. 4, 381-384.10.Choi CQ. Cell-O": Induced Pluripotent Stem Cells Fall Short of Potential Found in Embryonic Version. 2010. Scienti#c Ameri-can. 11.Rando TA. Stem cells, ageing and the quest for immortality. 2006. Nature. 441, 1080-1086. 12.Rosenthal, Nadia. Stem Cells and the End of Ag-ing. 2008. !e Howard Hughes Medical Institute Lecture Series.. 13.Carlson ME, Suetta C, Conboy MJ, Aagaard P, Mackey A, Kjaer M, and Conboy I. Molecular aging and rejuvenation of hu-man muscle stem cells. 2009. EMBO Molecular Medicine. 1, 381-391. 14.Drize N, Chertkov J, Sadovnikova E, Tiessen S, and Zander A. Long-Term Maintenance of Hematopoiesis in Irradiated Mice by Retrovirally Transduced Peripheral Blood Stem Cells. 1997. Blood. 89, 1811-1817. 15.McDonald, Kim. Biologists Image Birth of Blood-Form-

21

Reviews


ing, Stem Cells in Embryo. 2010. UC San Diego News Center. . 16.Butler JM, Nolan DJ, Vertes EL, Varnum-Finney B, Kobayashi H, Hooper AT, Seandel M, Shido K, White IA, Kobayashi M, Witte L, May C, Shawber C, Kimura Y, Kitajewski J, Rosenwaks Z, Bernstein ID, and Ra#i S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. 2010. Cell Stem Cell. 6, 251-264. 17.Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton D, and Benvenisty N. E"ects of eight growth factors on the di"erentia-tion of cells derived from human embryonic stem cells. 2000. Proc. Natl. Acad. Sci. U.S.A. 97, 1130711312.18.Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, and McKay R. Di"erentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets. 2001. Science. 292, 13891394.19.Brennard K and Melton D. Slow and steady is the key to -cell replication. 2009. J Cell Mol Med. 3, 472-487.20.Anthony Atala Lab. Engineering Pancreatic Beta Cells. Wake Forest University Center of Regenerative Medicine. .21. Furth ME and Atala A. Stem cell sources to treat diabetes. 2009. J Cell Biochem. 106, 507-511.22.Shrivastava D and Ivey KN. Potential of stem-cell-based thera-pies for heart disease. 2006. Nature. 441, 1097-1099.23.Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. 2003. Cell. 114, 763776. 24. Murry CE, Reinecke H, and Pabon LM. Regeneration gaps: observations on stem cells and cardiac repair. 2006. J Am Coll Car-diol. 47, 1777-1785. 25. Mathur A and Martin JF. Stem cells and repair of the heart. 2004. Lancet. 364, 183192.26. Cohen S and Leor J. Rebuilding Broken Hearts. 2004. Scien-ti#c American. 45-51.27. Nelson TJ, Fernandez AM, Yamada S, Terzic CP, Ikeda Y, and Terzic A. Repair of Acute Myocardial Infarction by Human Stem-ness Factors Induced Pluripotent Stem Cells. 2009. Circulation. 120, 408-416.28. Doris Taylor Lab. University of Minnessota Stem Cell Insti-tute. .29. Lab-grown bladders a milestone. 2006. BBC News. .30. Anthony Atala Lab. Engineering a Blood Vessel. Wake For-est University Center of Regenerative Medicine. .31. Lindvall O and Kokaia Z. Stem cells for the treatment of neu-rological disorders. 2006. Nature. 441, 1094-1096.32. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sdhof TC, and Wernig M. Direct conversion of #broblasts to functional neu-rons by de#ned factors. 2010. Nature. 463, 1035-1041. 33. Cogner K. Dramatic transformation: Researchers directly turn mouse skin cells into neurons, skipping IPS stage. 2010. Wernig Lab. .34. X-Cell Institute of Regenerative Medicine. .

35. Tong A and Kalb L. $62 million UC Davis center puts Sac-ramento at hub of stem cell research. 2010. !e Sacramento Bee..

Reviews


Seeing at the Atomic Level.DUWLN.XPDUDJXUXSDUDQ:DUG0HOYLOOH+LJK6FKRRO

Imaging techniques have and continue to foster our scienti#c understanding. From the invention of the light microscope, which led to the discovery of cells, to the use of X-ray crystallography to elucidate the structure of proteins and nucleic acids, imaging has allowed for our insight into biology to increase. Better imaging techniques and instruments continue to be developed in order to overcome the constraints of the human eye. While the resolution of a light microscope is limited by the wavelength of illuminating light, an electron microscope gives us a much greater resolution de-pending on the voltage applied [1]. For its part, the Atomic Force Microscope (AFM), a scanning instrument, may allow us to visual-ize the individual atoms of a molecule, and obtain results similar to the ball and stick models seen in chemistry textbooks. In fact, recent work by IBM scientist Leo Gross and his colleagues dem-onstrates how modifying the atomic microscopes tip apex yields an atomic resolution image of pentacene [2].

!e Atomic Force Microscope was originally created in 1986 to overcome the drawbacks of the Scanning Tunneling Microscope (STM). Since the STM can only create images of the samples placed on a conducting or semi-conducting surface, the types of samples that can be viewed are severely limited. !e AFM, howev-er, has the ability to create images on any kind of surface including polymer, biological and ceramic substrates. Before the #ndings of Gross et al., atomic force microscopy was used to resolve the struc-ture of separate atoms, but not that of atoms within an adsorbed molecule on a surface [2]. !e primary reason why the latter could not be accomplished was that the tunneling current was primarily sensitive to the local electron density of states close to the Fermi level [2]. A tunneling current is created when the tip of the STM

is close enough to the sample, and then an applied voltage between the tip and the sample causes the electrons to tunnel through the junction. !is current is dependent (exponentially) on the width of the electron junction. !is sensitivity can be used to create an image of atomic resolution [2]. When working at an atomic level, the term Fermi Level is constantly used in order to describe the amount of electron energy levels at absolute zero. Electrons, which are known as Fermions, cannot exist at the same energy states ac-cording to Paulis exclusion principle. At absolute zero these fer-mions try to all exist at the lowest energy level, which creates a sea of electrons. !e Fermi level occurs at absolute zero where no electrons are able to reach a higher energy level [3].

!e key to resolving the problem caused by the sensitivity of the tunneling current was to terminate the tip of the microscopes probe with a well-known and suitable molecule and understand that Pauli repulsion was the force that tampered with the image

Figures 1, 2. 7KHEDVLFSULQFLSOHEHKLQG$)0FDQEHXQGHUVWRRGZLWKWKHKHOSRIWKH$)0EORFNGLDJUDP>@(bottom left)DQGWKH9DQGHU:DDOVIRUFHGLDJUDP>@(above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c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

23

Reviews


Hyperphysics. Georgia State U, n.d. Web. 12 Nov. 2009. .4. Voice of Progress. Atomic Force Microscope (AFM). Voice of Progress. N.p., 31 Mar. 2009. Web. 8 Nov. 2010 .5. Nanoscience Education. Atomic Force Microscopy. nano Sci-ence Instruments. nano Science Instruments, 2008. Web. 2 Nov. 2009. .6. Galloway Group. Atomic Force Microscopy: A Guide to Un-derstanding and Using the AFM . 2004. 7. Nanotechnology. Scientists Image Anatomy of a Molecule using Noncontact Atomic Force Microscopy. Nanontechnology. AZoNetwork Sites, 28 Aug. 2009. Web. 1 Nov. 2009.

(the Van der Waals and electrostatic forces were found to con-tribute to background of the image rather than the actual image itself ). Pentacene, a well-studied polycyclic hydrocarbon, was the molecule the Gross group chose to investigate. Previous scanning tunneling microscopy tests on pentacene had found that resolving this molecule on metals such as Cu and thin #lm insulators such as NaCl was di%cult to accomplish using this technique. !is dif-#culty arose because STM imaging was a"ected by the density of the states when at the Fermi level (Energy of electrons within a semi-conductor) [2].

In order to create a high-resolution image of an atom within a molecule with AFM, Gross et al. had to operate within a short range of forces while simultaneously using, preferably, a sti" can-tilever with oscillation amplitudes of about 1 [2]. In order to successfully operate at even lower amplitudes such as 0.2 , it was necessary for the tuning fork to express a resonance frequency of 23,165 Hz. In addition to this setup, a carbon monoxide termi-nated tip was placed at the apex of the probe and resulted in the increased resolution of the image. !e idea for modifying the tip came from a similar procedure used on STMs that produced en-hanced resolution. By exploring this idea, Leo Gross et al. were able to create a cleaner and crisper image with higher resolution. !e tips were also modi#ed with Cl, pentacene, and metals such as Ag, Au, and Cu. Out of its (CO) #ve competitors, Cl gave an im-age with the next best resolution. Unlike the CO tip; however, the Cl tip produced an image that had a smaller set of benzene rings as well as less pronounced hollow sites above the minima [2].

Leo Gross, et al. concluded that Non-Contact Atomic Force Microscopy could indeed resolve the structure of atoms within molecules, if and only if the AFM entered the realm of repulsive forces [2]. At 1.2 , it was observed that the strength of repulsive forces reached maxima, which, therefore, provided the best con-trast and lateral resolution. Overall, the use of a CO terminated tip with AFM resulted in images that were crisp, clean, and informa-tive. Leo Gross et al. point out that the uses of such a discovery include, but are not limited to, analyzing catalysts, investigating single-electron transport chains and metal-molecule systems on the atomic scale [2].

Being able to visualize a molecule at this level may result in obtaining greater insight into the electronic and chemical proper-ties of any molecule at the atomic scale [7]. In fact, the Gross team and other IBM scientists hope to be able to understand electron transport through molecular networks and use this information to build smaller, faster and more energy-e%cient computing compo-nents [7]. !us, not only is the work of Leo Gross et al. notable for being the #rst time the chemical backbone of a molecule was imaged, but also for the profound impact that their results may have in nanotechnology.

5HIHUHQFHV

1. Karp, Gerald. Techniques in Cell and Molecular Biology. Cell and Molecular Biology: Fifth Edition. pp. 734-735. John Wiley & Sons, Inc., Hoboken, NJ. 2. Leo Gross et al. !e Chemical Structure of a Molecule Resolved by Atomic Force Microscopy. Science, Vol 325, 1110-1114, 2009. 3. Georgia State University (Physics Department). Fermi Level.

Perspectives


Each year thousands of students enter college with the hope of pur-suing medicine. However, a signi#cant percentage of these students never become physicians. !e latter is often the result of the immense number of premedical requirements that undergraduates must satisfy. In addi-tion to this intense workload, many of the premedical students who do master the requirements are often not admitted to medical schools be-cause of Medical College Examination Test (MCAT) score cuto"s and/or the limited number of #rst-year positions available [1]. For example, there were 42,269 applicants for medical school this year alone with only 18,390 #rst-year slots available [2]. It is no secret that the undergraduate years serve as a weeding out process for medical schools. At times, even some of the most successful students do not gain admission into medical school. !us, it is apparent that the road to becoming a doctor in the United States is one fraught with a variety of challenges.

7KH2ULJLQVRI3UHPHGLFDO5HTXLUHPHQWV

In the 19th century, the majority of medical institutions in the United States had very few formal premedical requirements. !e common belief was that men aspiring to practice medicine simply needed to have a gen-eral understanding of the sciences and thus essentially anyone with the means to a"ord medical school could study medicine [3]. However, in the early 20th century the American Medical Association (AMA) created a council to standardize premedical requirements for entrance into medical schools and to restructure US medical education as a whole. !e proposed premedical requirements were formalized and enumerated in Abraham Flexners 1910 report, Medical Education in the United States and Cana-da. As a result of Flexners report and work by the AMA council, premed-ical requirements necessitating competencies in biology, classical physics, and organic chemistry were #nally set in the 1930s. With the exception of the addition of a calculus requirement and an additional semester of organic chemistry, these requirements remain virtually unchanged. Con-sequently, as one might expect, there has been much disagreement in the past century over the purported bene#ts of the premedical curriculum, since many feel that the premedical requirements fail to achieve the in-tended goals.

Whats Wrong With the Current Premedical 5HTXLUHPHQWV"

!ough almost a century has passed since the Flexner report, it ap-pears that we are not any closer to understanding what may or may not be wrong with premedical education [4]. !e intended goals of the pre-medical curriculum are to give students a broad education so as to ulti-mately shape them as physicians who are able to reason morally and as-sess problems analytically and critically. However, many medical educators fervently argue that these goals are rarely achieved. Much of the criticism

of the current premedical curriculum is situated around its rigid empha-sis on the basic sciences, for many feel that this produces students who become narrowly focused and excessively concerned with grades. Some individuals favor the current curriculum because they see it as an e"ective way to thin out the medical school applicant pool. Nevertheless, many medical educators still feel that several of the courses that satisfy premedi-cal requirements do not truly enhance undergraduate premedical training. !ey argue that these courses are narrowly focused, for they often neglect to present any medical relevance [5]. Medical educators contend that pre-medical college courses mainly prepare students for material covered on standardized tests and thus cater to the MCAT. Consequently, there is now a call for undergraduate institutions to create premedical courses that will prepare students more adequately, by focusing on human biological principles and areas of science relevant to medicine [5].

In addition to this, many medical educators think that because of the narrow focus of premedical courses students often fail to grasp the cross-disciplinary nature of modern science. It is apparent that because we are complex organisms we are comprised of discrete systems, which are intri-cately linked within the body and modi#ed profoundly by external in$u-ences [5]. Consequently, many individuals argue that premedical courses should be taught in a manner that truly re$ects this interconnectivity. !is can be accomplished by transcending the old-fashioned compartmental-ization of undergraduate and medical school departments.

Most premedical curriculum reforms only examine the undergradu-ate level; however, many medical educators insist that revamping the cur-riculum alone is simply not enough. !ey argue that for one to truly assess and improve the issues with the premedical curriculum it is essential to reexamine the current structure of the medical system as a whole. Medical educators also stress that there needs to be more communication between undergraduate institutions and medical schools, for there is an evident disconnect between the sort of premedical preparation medical school faculty desire and the preparation that the current premedical curriculum actually provides. As a result, in the preclinical years medical school faculty often devote an extraordinary amount of time to elementary biochemistry and cell biology course material [5]. Moreover, medical school instructors argue that by neglecting to adequately address biologically relevant mate-rial, premedical science courses fail to prepare students for the rigor of preclinical course work. Medical educators also believe that many of the issues with the premedical curriculum stem from the current structure of the MCAT, for they argue that premedical science courses mainly prepare students for this exam. Consequently, they contend that by the end of undergraduate education most premedical students can only master the material covered on the MCAT.

Currently, premedical students are required to take calculus, phys-ics, and chemistry courses that mainly cover material presented on the MCAT, but rarely address biologically relevant material. !us, many med-ical educators believe that while it is necessary for premedical students

Revamping the Premedical Curriculum: A Move from Required Courses to Comprehensive Competencies)D\H0DULH9DVVHO

25

Perspectives


across the country to discuss ways to improve the current premedical re-quirements. A major goal of these meetings was to devise a set of skills, which premed students would need to satisfy, that accurately re$ect the multidisciplinary nature of modern medicine. !e report, Scienti#c Foundations for Future Physicians, published in June by the AAMC and Howard Hughes Medical Institute (HHMI) lists 16 competency skills, 8 of which articulate the science skills that medical students must gain before they leave medical school. !e remaining 8 competencies are a checklist of skills that premedical students would need to satisfy in place of the traditional required premedical science and mathematics courses. !e competencies are:

1. Apply quantitative reasoning and appropriate mathematics to describe or explain phenomena in the natural world.2. Demonstrate understanding of the process of scienti#c inquiry, and explain how scienti#c knowledge is discovered and validated.3. Demonstrate knowledge of basics principles and their applications of living systems.4. Demonstrate knowledge of basic principles of chemistry and some of their applications to the understanding of living systems.5. Demonstrate knowledge of how biomolecules contribute to the struc-ture and function of cells.6. Apply understanding of how molecular and cell assemblies, organs, and organisms develops structure and carry out function.7. Explain how organisms sense and control their environment and how they respond to external change.8. Demonstrate an understanding of how the organizing principle of evolution by natural selection explains the diversity of life. [9]

!is checklist aims to restructure the current premedical curriculum by moving away from a rigid set of required courses, thus giving students the option to study a wid

Documents

Young Investigators Spring 2010 Issue