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March 2016 | Vol. 6 No. 3

Approved for public release, distribution is unlimited

Baiting Mosquitoes

Revolutionizing Protein Design

Milestones for Synthetic Biology

A Cocktail of Biology

Lead DoD science and

technology to anticipate,

defend and safeguard

against chemical and

biological threats for the

warfighter and the nation.

DEFENSE THREAT REDUCTION AGENCY & USSTRATCOM Center for Combating WMD & Standing Joint Force Headquarters-Elimination

J9 Research and Development Directorate Chemical and Biological Technologies

8725 John J Kingman Road, Stop 6201, Fort Belvoir, VA 22060

www.dtra.mil

Handle: @cbdstconference

Keyword: DTRA

Channel: doddtra

On the cover:U.S. Navy Petty Officer 1st Class James Bowes, Camp Lemonnier’s expeditionary medical facility senior preventive-medicine technician, analyzes mosquitoes to help determine the risk of mosquito-borne diseases. All mosquito samples collected from Camp Lemonnier’s mosquito-control program are sent to the U.S. Army Public Health Command laboratory in Stuttgart, Germany, where researchers test the samples for species-verification and viruses. (U.S. Navy photo by Petty Officer 1st Class Tom Ouellette/Released)

On the back cover: Puerto Rico Army National Guard Medical Command provide Citizen-Soldiers with seasonal influenza vaccines. Here Captain Juan Rosado, 101st Troop Command S-4, receives his annual flu shot from PRNG MEDCOM personnel. (U.S. Army photo, Released by Staff Sgt. Joseph Rivera-Reboliedo)

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Deployed warfighters have more challenges than just fighting adversaries. Environmental conditions, such as the weather, terrain and vector-borne diseases all pose additional threats. Collaborative efforts between the Defense Threat Reduction Agency’s Joint Science and Technology Office and Sandia National Laboratories are focusing on new methodologies of vector surveillance to detect emerging infections in local mosquito populations and the development of a near real-time recording system to combat these threats.

This innovative research will provide warfighters with more information on mosquito-borne diseases in areas of deployment allowing for future vector control. Additionally, it could be utilized in the public sector to reduce manpower and turnaround time for general mosquito spraying operations in urban areas. Current approaches to vector surveillance are low-tech sample collectors that require work to be done manually and do not produce results for one to two weeks.

JSTO and Sandia National Laboratories are developing a smart-trap mosquito surveillance system that can be autonomous for one month and will wirelessly upload the results to the Biosurveillance Ecosystem (BSVE), a JSTO-developed, cloud-based software application that allows users to identify and quickly respond to public health concerns. The team is collaborating with the University of California to evaluate various liquid sugar baits that will lure mosquitoes to a small feeding station, called the “bait cave.” While feeding in the bait cave, the mosquitoes will deposit their saliva, which will contain any viruses they may be carrying.

To test for multiple viruses at once, the system runs loop-mediated isothermal amplification (LAMP). LAMP is an amplification process similar to polymerase chain reaction, but differs in that it performs the reaction at a constant

temperature instead of cycling through various ones. This allows for lower power requirements for the instrument and makes it more versatile for field operations. The box will remain dormant for the majority of the day, only turning on via battery, once a day to perform the analysis and upload results to the BSVE.

The current effort is developing the multiplex evaluation for West Nile, St. Louis Encephalitis and Western Equine Encephalitis viruses as they are all found in the vector Culex tarsalis and attracted to sugar baits.

Box development should be completed by the summer 2016 with field-testing beginning mid to late summer near the Salton Sea in Southern California. This smarter vector surveillance device will allow decision makers to understand the active diseases within a region and appropriately vaccinate and equip warfighters before deployment.

FOR SMARTER VECTOR SURVEILLANCE

What’s Your Vector Victor?

POC: Ms. Kathleen Quinn, kathleen.j.quinn3.civ@mail.mil

A prototype of the smart-trap surveillance system. Image provided by Sandia National Laboratories.

SME @ email address4

Of the possible patterns in protein molecules, only a fraction occur naturally, leaving room for scientists to fill functional gaps. This includes the production of an entire new class of materials patterned with atomic level accuracy that hold promise for support of warfighter needs.

In a project managed by Dr. Ilya Elashvili from the Defense Threat Reduction Agency’s Joint Science and Technology Office, researchers in Dr. David Baker’s lab at the University of Washington have reported several major advances in the computational design and construction of new proteins from scratch that possess desired characteristics.

Just as the manufacturing industry was revolutionized by creating interchangeable parts designed to precise specifications, modular proteins have the possibility to revolutionize the future of biotechnology. In a Nature article titled “Exploring the Repeat Protein Universe Through Computational Protein Design,” Dr. Baker’s team reported

building a series of novel repeat proteins with a broad range of geometries that are comparable to structural beams in manufacturing. The team designed and built robust structures with sequences unrelated to known repeat proteins with atomic level accuracy that were highly stable both chemically and thermally.

For example, in contrast to almost all native proteins, more than half (53 out of 83) of the designs were found to be monomeric proteins that were stable up to 95°C and four of the six designs tested did not denature at guanidine hydrochloride concentrations up to 7.5 mol/L. The ability to design computationally, highly stable proteins to precise specification is an enabling technology for rapid response to emergent military threat agents.

Repeat proteins can form very large binding surfaces using only a short amino acid sequence that is repeated in tandem. The computer designed repeat sequence

Similar to the steam engine revolutionizing the speed of manufacturing during the industrial revolution, new breakthroughs in computational protein design are revolutionizing warfighter countermeasures against chemical and biological threats. Proteins, composed of linear chains of amino acids, are miniature machines responsible for most critical functions in living organisms. The sequence of amino acids determines the shape of the protein, which is essential to carrying out its biological function.

Triosephosphate isomerase barrel blueprint: The eight-fold symmetric TIM-barrel protein design paradigm is illustrated graphically in the form of a blueprint with the zeros and ones representing computer calculations for a real protein based on the Rosetta design specification for the TIM-barrel. This class of protein folds in nature carries out a wide variety of the enzyme reaction types. Computational design of proteins now allows researchers to build entire new families of these TIM-barrel proteins. Image courtesy of Dr. David Baker and Dr. Po-Ssu Huang, University of Washington.

Full Steam Ahead:

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enables designers to make proteins that take on shapes which are straight, or twist with defined pitch, and even bend back on themselves to form nano-rings. Dr. Baker’s team, in collaboration with Dr. Philip Bradley’s group at the Fred Hutchinson Cancer Research Center, reported this in a Nature article titled “Rational Design of α-Helical Tandem Repeat Proteins with Closed Architectures.” These proteins open a wide variety of new possibilities for biomolecular engineering such as the ability to create new enzymes, organized catalytic complexes, protein materials and biosensors.

In another article in eLIFE titled “Precise Assembly of Complex Beta Sheet Topologies from De Novo Designed Building Blocks,” the Baker researchers demonstrated successful design of larger beta sheet domains by recombining smaller independently folded beta sheet proteins. They demonstrated that protein topologies with six- and seven-stranded beta sheets can be designed by inserting a de novo, designed from scratch, beta sheet containing protein into another, so the two beta sheets are merged to form a single extended sheet.

In addition to their robust design methods in made-to-specification repeat proteins and enlarging beta sheet domains, the Baker group also expanded their

fundamental understanding of scaffolds used in biocatalysts. Baker lab postdoctoral fellow Po-Ssu Huang, together with Dr. Birte Höcker at the Max Planck Institute for Developmental Biology (Tübingen, Germany)

turned their attention to the (beta/alpha)8 topology — commonly known as the triosephosphate isomerase barrel, or TIM-barrel fold, which is one of the most widely used enzyme folds in biology.

Enzymes that adopt this fold are responsible for five of the six enzyme class reaction types. It has been considered one of the most important folds for designing novel catalysts. For nearly three decades, multiple attempts have been made to design the barrel fold, but none demonstrated success. The researchers were the first to achieve this goal, reported in the Nature Chemical Biology article titled “De Novo Design of a Four-Fold Symmetric TIM-

Barrel Protein with Atomic-Level Accuracy.” The success of understanding TIM-barrel fold enables technologies for designing catalysts that can inactivate chemical agents which threaten warfighters in the battlefield. Particularly, many native TIM-barrel enzymes interact with phosphate-containing compounds, and this feature alone has significant implications in designing new proteins that can sense and breakdown organophosphate agents, such as the nerve agents sarin and VX.

Portrait of repeat protein possibilities: The repeat protein design paradigm is illustrated in the central graphic as a specification for repeated alpha helical segments (yellow and blue) connected with caps and loops (orange) with specified direction of writhe, rise and angle. Repeat proteins are critical to life at its basic level for molecular recognition, cell signaling and tethering chemical messengers. Repeat proteins are inspiring ideas for molecular inventions to perform microscale jobs in medical, environmental and industrial arenas. A tiny fraction of the possibilities for repeat proteins are shown to represent the vast diversity of repeat protein structures that are now accessible. Illustration by the University of Washington.

Revolutionizing Protein Design for Warfighters

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for logy

Through continued partnerships with the intelligence community and U.S. Military Service Academies, JSTO is focusing on responsible development of new countermeasures enabled by synthetic biology. In 2014 the Assistant Secretary of Defense for Research and Engineering emphasized the importance of DoD investments in synthetic biology for specialty materials, sensing and chemical and biological defense. Service members lacked a deep understanding of synthetic biology and the ASD (R&E) recommended instituting bioengineering programs at each service academy.

To support this initiative, JSTO launched the Syn Bio Academies Challenge, a pilot program at the U.S. Military Academy at West Point and the U.S. Naval Academy at Annapolis, where cadets and midshipmen compete for an annual prize. This competition, managed by Dr. Brian Pate of DTRA, is administered in conjunction with the 2016 International Genetically Engineering Machine

(IGEM) Competition. This premier global synthetic biology student conference focuses on education, competition and advancement of synthetic biology open collaboration.

Under the Service Academy Research Initiative, DTRA-sponsored projects have incorporated an increased use and investigation of synthetic biology approaches, with the goal of increasing preparedness of future warfighters to anticipate and perform appropriately in their military environments.

JSTO also fosters national expertise in synthetic biology through its broader research investments focused on countermeasures to chemical and biological weapons, driven by DoD requirements as well as DTRA-identified knowledge gaps.

For example, under DTRA’s chemical countermeasures program, a project managed by Dr. Pate developed a targeted

Gene editing allows scientists to combine wooly mammoth and elephant DNA, engineer pig organs for human transplants and create malaria-resistant mosquitoes that can pass their resistance to offspring. While gene editing could help eradicate some of the world’s greatest global health concerns, it also has the potential to be a weapon of mass destruction.

The U.S. Director of National Intelligence, James Clapper, recently named gene editing as a priority weapon of mass destruction threat; the only biotechnology included in the worldwide threat assessment report, due to recent advancements within synthetic biology. As such, the Defense Threat Reduction Agency’s Joint Science and Technology Office is preparing next generation warfighters to be leaders in this emerging field.

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Liposome shell Targeting moiety

Targeted Nanoparticle

Payload

Customized for each payload

DNAPlasmids

nanodelivery platform. The platform consists of a liposomal shell, a single chain of antibody fragments as targeting moieties, and a payload that includes

DNA plasmids coding for proteins active in scavenging nerve agents. In preliminary trials, this synthetic biology approach was capable of upregulating the endogenous expression of nerve agent scavenging protein within the brain.

Other DTRA-managed projects with a significant synthetic biology component include efforts focused on manipulating simple organisms or ex-vivo genetic circuits. These circuits impart the ability to recognize, signal and defeat chemical or biological threats with built-in “kill switches.” The “kill switches” deactivate these genetically-modified organisms when they are no longer useful. In pursuing responsible applications of synthetic biology, DTRA is both mitigating current and future threats as well as enhancing relevant national scientific capacity.

Despite Russia’s signatory status to the Biological and Toxin Weapons Convention, which bans development of biological weapons, Russian Prime Minister Vladimir Putin stated in 2012 that, “in the more distant future, weapons systems based on new principals (beam, geophysical, wave, genetic, and other technology) will be developed. All this will, in addition to nuclear weapons, provide entirely new instruments for achieving political and strategic goals. Such hi-tech weapons systems will be comparable in effect to nuclear weapons but will be more “acceptable” in terms of political and military ideology.”

Such statements underscore the critical nature of DTRA’s work in fostering U.S. military and civilian scientific capacity in the field of synthetic biology, as well as leading the pursuit of responsible applications of this important technology to countering weapons of mass destruction.

Targeted nanodelivery platform developed for chemical defense applications under DTRA management by SynerGene Therapeutics Inc. Image courtesy of SynerGene Therapeutics Inc.

POC: Dr. Brian Pate; brian.d.pate.civ@mail.mil

SME @ email address8

A Cocktail of Biology: Shakin’ up the Space

with Synthetic Ingredients

Consider genetic engineering, molecular biology, nanotechnology, biochemistry and systems biology. What do they all have in common? When you combine the application-orientated sides of these diverse scientific disciplines and throw in a forward engineering approach adopted from mechanical and electronic engineering, you get synthetic biology. This multidisciplined approach enables new medical and environmental diagnostic tools to protect the warfighter.

Synthetic biology is responsible for new classes of inexpensive, rapidly deployable diagnostic devices and is moving towards near real-time surveillance of multiple pathological conditions to protect the warfighter from chemical and biological threats. An article by Defense Threat Reduction Agency-funded researchers, Drs. Shimyn Slomovic, Keith Pardee and Jim Collins, from the Massachusetts Institute of Technology and Harvard University, highlights several milestones and offers insight into where the field is headed. The article, “Synthetic Biology Devices for In Vitro and In

Vivo Diagnostics,” marks the 100th anniversary of the Proceedings of the National Academy of Sciences and focuses on the evolution of biosensing.

Environmental and whole-cell biosensing

From a historical perspective, biosensing is not a new concept. People have used canaries in coalmines and tracking dogs for centuries to sense environmental hazards. Animal sentinels were scaled down to single living cells in the years leading up to the onset of synthetic biology as a field. The natural biochemical or enzymatic pathways of these first “whole-cell biosensors” were exploited to evaluate the environment.

For example, the photosynthetic activity of electrode-mounted cyanobacteria produced a current that disrupted herbicides in water, alerting to the presence of the chemicals. Such hybrid bio-electrical devices were gradually phased out as scientists introduced bioluminescence-producing gene cassettes into microorganisms, converting them into purpose-built

analyte sensors with their own signal output components. However, as microorganisms are progressively modified to include synthetic gene circuitry that allows them to detect arsenic, hydrocarbon pollutants and other compounds, the authors predict a revitalized integration of companion electronic components for rapid field deployment.

The Collin’s team also discusses several advances they made as the result of DTRA’s Joint Science and Technology funded project managed by Dr. Ilya Elashvili. Notably, they describe how bacterial cells can be programmed with synthetic gene circuitry that transforms them into living RNA-based biosensors, which they reported in the Cell article titled “Toehold Switches: De Novo-Designed Regulators of Gene Expression.”

In this paper, the scientists used clever computational algorithms to design RNA “toehold switches” that can be expressed in microorganisms. These RNA-based sensors stem from conventional translational

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A. Biosensing the environment

Animal sentinels

Tumor imaging Prosthetic networks

Microbial sentinels

Delivery “nanobots”

Native and engineered whole-cell biosensors

Phage diagnostics Paper-based

diagnostics Wearable diagnostics?

B. In vitro diagnostics

C. In vivo diagnostics

riboregulators, where a toehold hairpin structure complementary to the target is engineered at the upstream of reporter protein mRNA’s ribosomal binding site (RBS). The hairpin blocks the RBS binding to ribosome, thereby preventing the initiation of reporter protein synthesis. Upon encountering the complementary target (“trigger”) RNA sequence, the hairpin is unzipped (through Watson-Crick base pairing) and the mRNA binding to the RBS is unblocked.

As a consequence, the target RNA sensing is translated to yield the production of fluorescent or colorimetric reporter proteins. The rational de novo, designed from scratch, design of these molecular switches shows great potential for intracellular diagnostics related to disease-specific RNAs. The article proceeds to describe how biosensing machinery has been taken outside of the cell and is currently fulfilling a range of in vitro diagnostic applications.

In vitro diagnostics

Engineered bacteriophages are trailblazing the in vitro diagnostics space. With their natural, strain-specific sensing capabilities, bacteriophages equipped with reporter genes can be deployed to detect, infect and transform pathogens into self-informants that alert of their own presence. These bacterial viruses are inexpensive to make, can be optimized through directed evolution for enhanced target specificity, and are currently in use as pathogen sensors both in clinical settings and in the

food industry. Currently the Centers for Disease Control and Prevention uses an engineered plague diagnostic phage, ϕA1122, to detect Yersinia pestis (the etiologic agent of the bubonic plague) in clinical samples in just two hours.

While engineered bacteriophages have begun to take synthetic biology out of the lab, paper-based diagnostics force synthetic biology out of the cell. Cell-free expression systems are commercially available and scientists have also streamlined the production of home-brewed lysates, making it cheaper to translate and screen proteins in vitro. The authors discuss how the same JSTO-funded effort accelerated paper-based systems by freeze-drying coupled transcription/translation reactions onto paper.

In this regard, they cite their

accomplishments published in the Cell article, “Paper-Based Synthetic Gene Networks.” Here, the authors demonstrate that synthetic gene circuits encoding diagnostic RNA toehold sensors can be freeze-dried in the cocktail as well, and they remain stable for months on end and poised for action until hydrated with a clinical sample or water. For example, this technology allowed Collin’s team to construct 24 different Ebola sensors in less than 12 hours with the DNA input costing only $21 per sensor.

The Collins group describes how this technique was used for sensing both small (e.g., glucose) and large molecules (e.g., Ebola virus RNA), including strain-specific Ebola virus sensors. The authors posit that paper-based systems have opened the door to a robust,

Figure courtesy of Dr. James Collins, Massachusetts Institute of Technology.

Synthetic biology’s contribution to environmental, in vitro and in vivo diagnostics.A. Environmental biosensing began with animal sentinels and led to bacterial sensors coupled to electronic equipment and genetically modified microorganisms. B. Engineered bacteriophages take circuitry out of the cell and paper-based systems bring it to the field. Diagnostics may one day be embedded in wearables. C. Both microbial and mammalian cells are now used to evaluate the in vivo space: with bacteria for tumor imaging, prosthetic networks to correct deficiencies, sentinels that record drug exposure in the gut and DNA origami-based nanobots that deliver circuits to specific diseased cells.

SME @ email address10 POC: Dr. Ilya Elashvili, ilya.elashvili.civ@mail.mil

cheap, mobile and safe means to conduct diagnostic biosensing in low-resource conditions. This new ability has wide applicability to both deployed warfighters and to resource-poor countries.

In vivo diagnostics

Synthetic biology initially aspired to reproduce simple mechanical switches in genetic form, drawing on design principles from mechanical and electrical engineering. Early version genetic toggle switches later grew into increasingly complex circuits that could compute Boolean logic and even facilitate genetic memory. With these advanced circuit design and implementation techniques, researchers realized the potential in using engineered microorganisms to sense the “innerspace” of the human body for real-time diagnostic monitoring.

Collins and his team provide an interesting example of sentinel E. coli engineered with sophisticated toggle–switch circuitry that allows them to patrol the mouse gut, record drug exposure, and report their findings in stool samples. Genetically modified bacteria have also been applied to home in on cancerous growth and relay spatial information via bioluminescence. In the future, such sentinel microorganisms could be critical for early diagnosis and tracking of metastasis, drastically improving the success rates of targeted treatments.

Mammalian cells are also beginning to play important diagnostic and therapeutic roles, carrying out actions

that bolster natural biological processes and fix deficient ones. One example given for diagnostic mammalian biosensors describes human cells that were engineered to express the bacterial luxCDABE gene cassette, which allows them to produce a bioluminescent signal following their subcutaneous injection.

The authors also describe the groundbreaking cell-profiler system, in which human cells are equipped with synthetic circuits that allow them to assess the levels of microRNAs that mark specific cancer cells. These sentinels can pick cancer cells out of a “line-up” and either make them glow red or force them to self-destruct by evoking apoptosis. In perhaps the most exquisite example of the therapeutic potential of synthetic biology in mammalian cells, the article discusses a “prosthetic” gene network introduced into encapsulated human cells that are injected into a mouse model to fight gout and tumor lysis syndrome. These cells are able to restore urate homeostasis using genes borrowed from bacteria and fungi, demonstrating a prosthetic gene network concept that holds great potential for future use in humans.

The delivery of genetic constructs into specific cell types and tissues in the human body poses a challenge that is more formidable than drug delivery, as these genes must be expressed once they reach their destinations. Fortunately, researchers are developing an array of delivery means, and the authors detail several of these approaches.

“Nanobots” are origami DNA barrels that can be loaded with genetic content to be delivered to specific cell types whose surface receptor keys open locks found on the bot’s hinges.

For delivery to tumors, the authors envision the conjugation of nanobots to flagellated commensal bacteria whose self-propulsion and natural tumor-homing attributes could raft the bots to cancerous growth. In addition to adeno-associated viruses and self-assembled virus-like particles, which presently play a prominent role in delivery, biological vesicles have begun to attract much attention.

These lipid pockets can be produced from Gram-negative bacteria and even from exosomes secreted by mammalian cells. Such vesicles are loaded with genetic material and outfitted with cancer-specific antibodies that allow cell-specific delivery and expression of fluorescent reporters, toxins, and anti-cancer small interfering RNAs. The Collins team floats the intriguing prospect of biological vesicles that could be automatically loaded with the appropriate genetic material by “manufacturing” cell lines, which would drastically improve the efficiency and rate of their production.

Synthetic biology has come a long way since its inception, and with it, our ability to engineer novel biological functions, sense the natural environment, diagnose our ailments, and monitor our bodies’ internal environment. Biosensing has evolved from animal sentinels to synthetic gene circuits fixed on paper, allowing us to take diagnostics out of the lab and into the field and protect the warfighter. Bacterial, mammalia, and artificial cells are engineered to patrol our insides and inform us of disease-indicating perturbations, and in some cases, to dynamically respond and correct these negative states allowing strong medical counter measures for the warfighter, while having applicability to a wide-range of aliments the world faces.

A Cocktail of Biology ...(continued from Page 9)

SME @ email address 11POC: Dr. Ilya Elashvili, ilya.elashvili.civ@mail.mil

With this success, researchers now understand down to the residue level the roles of every amino acid in this designed scaffold; this designed protein is among one of the most well behaved TIM-barrels. This represents a new platform for designing future catalysts and biosensors for various small molecule chemical agents. Researchers anticipate extending this science to interact with proteinaceous agents through the various loops built on the barrel. This would essentially mimic antibodies, but with a larger footprint, having a significant impact in protecting warfighters from chemical and biological agents.

The options for engineering brand new protein materials through modular construction with atomic level precision have been thrown open. In addition to repeat proteins and TIM-barrels, the Baker group has also reported the precision sculpting of smaller de novo designed folds made to order, never before seen in nature. This progress was reported in Proceedings of the National Academy of Sciences in a paper titled “Control Over Overall Shape and Size in De Novo Designed Proteins.”

This paper further explains methods for systematically varying protein architecture inspired by nature controlling structural variation within the same fold. Finesse in protein design is needed to optimize designed proteins to take on exact shapes to perform specified functions. Baker graduate student Yu-Ru Lin, in collaboration with Dr. Nobuyasu Koga from the Institute for Molecular Science in Japan, led this research.

Future applications of atomic level accurate protein de novo design strategies with control over fold shape, size, twist, length, surface features, loop locations and several other architectural features will enable the simultaneous design of robust protein sequences for form and function. These advances in biotechnology could revolutionize computationally designed high-affinity binding proteins, enzymes and protein-based nanomaterials that meet specific needs for the warfighter in chemical and biological defense.

Revolutionizing Protein Design for Warfighters ...(continued from Page 5)

To learn more about DTRA CB, a

short book, Innovative Science for

Chemical and Biological Defense,

is offered online on Apple iBooks

and Google Play, free of charge.

Keyword: DTRA

Within the Defense Threat

Reduction Agency’s Research

and Development Directorate,

resides the Joint Science and

Technology Office for Chemical

and Biological Defense. This

publication highlights the

organization’s accomplishments

to protect warfighters and

citizens through the innovative

application of science and

technology research.

DTRA

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