Nanodevices built with DNA origami

Did you know DNA could be used for origami?

Not actual DNA origami.

Not actual DNA origami.

The precise control and organization of nanoscale devices has shown a great potential for ultimately creating “nano-devices” that can perform nanoscale biological measurements, deliver medicine in vivo, among many other applications. A recent article from Carlos E. Castro and colleauges from The Ohio State University demonstrates the use of DNA origami with programmable complex and reversible 1D, 2D and 3D motions.

By varying the DNA origami design, they were able to observe different mechanisms for the DNA origami’s 3D motion such as the crank-slider and four bar mechanism. The research team mainly utilized transmission electron microscopy (TEM) to follow the morphology changes as the origami moves.

DNAUsing a fluorescence quenching assay (attaching a fluorescent label on one arm and a quencher on the other), they have characterized the timescale of DNA origami motion. Overall, their group sees this technology as a “foundation for developing and characterizing a library of tunable DNA origami kinematic joints and using them in more complex controllable mechanisms similar to macroscopic machines, such as manipulators to control chemical reactions, transport biomolecules, or assemble nanoscale components in real time.”

 

Shown below are some of the videos showing the motions of the DNA origami that they have reported:

About the author: Herdeline Ann M. Ardoña is a third year graduate student at Johns Hopkins University Department of Chemistry, currently working in chemistry professor J.D. Tovar’s lab and co-advised by professor Hai-Quan Mao, in materials science and engineering.

Reference: Programmable motion of DNA origami mechanisms. (Proc. Natl. Acad. Sci. U.S.A., 2015, 112, 713-718)

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Are there problems with the peer-review process?

The pathway to publication is littered with checkpoints, reviews, and rejections. Before your paper is accepted it is read and reviewed with a few possible fates. It can be desk rejected by the editor and never reviewed or it can reach the reviewers who then decide the fate of the manuscript.

questionamarkwebSiler et al. investigated the effectiveness of the review process. They observed that top ranking journals overall have a very effective desk screening process where the best manuscripts are selected for review. However, there was one main fault; these top tier journals desk rejected the top cited manuscripts. This is likely due to the fact that their goal is to publish papers that are widely applicable and of interest to many people. This limits the ability of truly novel and exciting works to be published in these formats.

Overall, however, it was determined that the review process is helpful. Manuscripts that went to review overall had more citations than those desk rejected and resubmitted elsewhere. The results of this study were reassuring, and it was nice to see that at least a few scientists are looking into the effectiveness of the review process.

Link to article: http://www.pnas.org/content/112/2/360.abstract

About the author: Moriah Knight is a third year in the Johns Hopkins Department of Materials Science and Engineering working in Peter Searson’s lab.

Nano-bio lab course: MAPs and CD

Editor’s note: Over the next several days, we will share the student impressions of some of the techniques learned in INBT’s nano-bio laboratory course (670.621). These reports demonstrate the wide variety of techniques students trained at the Johns Hopkins Institute for NanoBioTechnology are expected to understand. Each technique is taught in a different affiliated faculty lab. More lab techniques to come.

Membrane Active Proteins (MAPs) and Circular Dichroism (CD) spectrography

During this lab, we learned a couple of techniques that I had not used before. First we synthesized liposomes and processed them, resulting in uniform liposome radius. Then we made a solution of membrane active proteins with aromatic amino acids so that their absorbance and emission could be measured.

CD_trans

circular dichroism (CD) spectrography

We ran the proteins through the fluorometer at varying wavelengths to create a profile of emission and absorbance of the protein. This was done also at varying pHs and at different liposome concentrations.

In theory the proteins should incorporate into the liposomes and there should be a change in the spectra as a result. During our lab time we had issues getting the desired results, but it was still informative on how to use the fluorometer and other new equipment. We found the spectra for two different proteins at two different pH values for each to see the effect that pH had on the emission/absorbance spectra.

We also preformed CD spectrography (circular dichroism) to determine the chirality of the proteins, that is, how are the specific molecules spatially arranged. Again the procedure did not work exactly as planned, but learning how to perform the measurement was informative, nonetheless.

About the author: Jackson DeStefano is a first year PhD candidate in the laboratory of Peter Searson, professor of materials science and engineering.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.
.

How firefly research helped gene therapy

Sometimes on a calm summer or fall night, one is able to observe the beautiful dance of blinking fireflies. Scientists began to explore mechanisms to describe this unique natural phenomenon as early as the late 1800’s. After a series of experiments with solutions at different temperature with ground up abdomens of fireflies, Raphael Dubois named the enzyme luciferase and the substrate luciferin that were the cause of the light-producing reaction (1).  But it wasn’t until recently in 1985 that scientists were able to clone the gene for luciferase and express it in bacteria to produce the luciferase.

firefly

Figure 1: Picture of firefly. Source: http://www.fireflyexperience.org/photos/

Once the gene was cloned, genetic researchers realized the importance of the findings and started to use it as a reporter gene for experimental gene therapy. Gene therapies involve transfection of new genetic material into the host’s DNA and can be applied not only for therapies for diseases of genetic origin, but can be used for cancer therapy and diagnostic purposes.

By incorporating the gene for luciferase along with the gene of interest, the Hai-Quan Mao lab in the Department of Materials Science and Engineering at Johns Hopkins University can detect whether or not their nanoparticles used for gene delivery have been successful simply by adding luciferin to the cells. If the gene transfer was successful, then the luciferase will act on the substrate luciferin to emit light.

Sources

1)     Fraga, Hugo. “Firefly luminescence: A historical perspective and recent developments.” Photochemical & Photobiological Sciences 7.2 (2008): 146-158.

About the author: John Hickey is a second year Biomedical Engineering PhD candidate in the Jon Schneck lab researching the use of different biomaterials for immunotherapies and microfluidics in identifying rare immune cells.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

 

O-GlcNAc: The Sweet Side of Epigenetics

In 1992 Edmond H. Fischer and Edwin G. Krebs won the Nobel Prize in Physiology or Medicine “for their discoveries concerning reversible protein phoshorylation as a biological regulatory mechanism.” Phosphorylation of proteins can essentially be thought of as the on/off switch that regulates protein activity inside of cells.

It became increasingly clear later on, however, that protein physiology was much more complex than regulation through just a simple on/off phosphorylation switch. It was eventually discovered by Johns Hopkins’ very own Dr. Gerald Hart that a very special sugar called N-acetylglucosamine (GlcNAc) can be added to the same places on proteins where phosphorylation often occurs. The addition of GlcNAc to these sites is now known as the O-GlcNAc modification. O-GlcNAc essentially serves as another layer of control over protein physiology by acting as a sort of “cap” that must be removed before a protein can be phosphorylated. In otherwords, phosphorylation and the O-GlcNAc modification cycle between each other to regulate how many important proteins behave. One amazing feature of the O-GlcNAc modification is the fact that it is performed by only two enzymes, OGT which adds it to proteins and OGA which removes it, and that’s it. This is in stark contrast to protein phosphorylation and dephosphorylation which needs hundreds of different enzymes to perform phosphorylation mechanics.

Fig 1.  Histones are modified by O-GlcNAc.

Fig 1. Histones are modified by O-GlcNAc.

To this day O-GlcNAc cycling remains an enigma, however, emerging evidence continues to mount that illustrates the very important physiological roles for O-GlcNAc. Two of some of the most important concepts within the realm of epigenetics are the modifications of histones and the methylation of DNA. It is now known that histones, which are proteins that help package DNA into the nucleus, are modified by O-GlcNAc 1 (fig 1.). The other major type of epigenetic regulation of gene expression– methylation of DNA–silences genes, but is also a reversible process. Proteins named TETs help to remove methyl groups on DNA to reverse this silencing. Recently it has also been shown that TETs have their activity regulated by O-GlcNAc 2. In otherwords, O-GlcNAc seems to have a very important role in regulating and interacting with two very important physiological mechanisms that write the epigenetic code.

Finally, glucose is most often thought of as fuel for the cell–and this is true–however, the substrate that is required to perform the O-GlcNAc modification (GlcNAc) happens to also be a byproduct of glucose metabolism. Major diseases such as cancer, diabetes, and Alzheimer’s are often associated with altered glucose metabolism and also have profound epigenetic changes. It is quite tempting, therefore, to postulate that O-GlcNAc may be the key that links environment, stress, nutrient availability, and metabolism to changes in epigenetics. Understanding carbohydrate metabolism and O-GlcNAc regulation of epigenetics may one day open new doors that will lead to breakthroughs in regenerative medicine, understanding embryological development, tissue engineering, and treating major diseases.

About the Author: Christopher Saeui is a fourth year Biomedical Engineering PhD student in the Kevin J. Yarema Laboratory for Cell and Carbohydrate Engineering studying the epigenetic and metabolic mechanisms that alter glycosylation in cancer.

References
1. Sakabe, K., Wang, Z. & Hart, G. W. Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code. Proc. Natl. Acad. Sci. U. S. A. 107, 19915-19920 (2010).
2. Shi, F. T. et al. Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells. J. Biol. Chem. 288, 20776-20784 (2013).

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Lab-on-a-chip helps understand cardiovascular disease

Cardiovascular disease is the leading cause of death in the United States accounts for over 600,000 mortalities and $400 billion dollars in health related costs annually. If we can study vascular behavior in an in vivo mimicking environment, we can better understand how the roadblocks of chemical inflammation, elevated blood pressure, plaque formation and blood vessel narrowing contribute to cardiovascular disease.

Endothelial colony forming cells with shear stress.

Endothelial colony forming cells with shear stress.

The circulatory system contains a dense network of blood vessels, which are the highways that sustain underlying tissue by mediating the transfer of nutrients and removal of waste. These highways are lined with mechanosensing endothelial cells (ECs) that direct the travel of sustenance contained within the blood by acting as selective barriers.

One hallmark behavior of ECs is their ability to align and elongate in response to blood flow generated from heart contraction. By recapitulating this scenario in the lab using microfluidic (lab-on-a-chip) systems, ECs can be cultured in dynamic environments that circumvent the limitations of traditional in vitro culture platforms.

About the author: Quinton Smith is a third year graduate student in the Department of Chemical and Biomolecular Engineering, studying the effects of physio-chemical cues governing stem cell behavior and maturation under the mentorship of Sharon Gerecht, associate professor. 

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Finding new tasks for the disruptive proteins in bee venom

When a bee stings you, it leaves its stinger within your body releasing a peptide toxin called melittin. Melittin is toxic to cells because it is able to insert and bind to the cell membrane, which destabilizes the membrane.

Melittin is able to interact with the hydrophobic lipid layer of the cell membrane by forming unique helixes where hydrogen bonding occurs between both peptides. Consequently, charged (polar) amino acid residues are generally not observed in such proteins and pH can change the binding affinity. Such proteins are called membrane active proteins (MAPs).

Now this is not good for your bee-stung cells, but researchers are looking to repurpose nature’s disruptive proteins as anti-microbial drugs, cancer therapeutics, and HIV drugs. Specifically, researchers in the Kalina Hristova lab in the Department of Materials Science and Engineering at Johns Hopkins are engineering proteins based off of the melittin protein.

Figure 1: Membrane Active Peptide Schematic (Source: http://ins.sjtu.edu.cn/people/jakob/)

Figure 1: Membrane Active Peptide Schematic (Source: http://ins.sjtu.edu.cn/people/jakob/)

The Hristova lab researchers study their developed MAPs by using lab-produced vesicles from phosphatidylcholine (the major component of a cell membrane). They use the natural fluorescence from tryptophan (which increases in a hydrophobic environment), and circular dichroism spectroscopy (which is able to detect the chiral structure of proteins) to verify the peptide’s interactions with the vesicles, and what affinity they will bind.

About the author: John Hickey is a second year Biomedical Engineering PhD candidate in the Jon Schneck lab researching the use of different biomaterials for immunotherapies and microfluidics in identifying rare immune cells.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Engineering bacteria for medical uses

According to the National Institutes of Health and Centers for Disease Control, drug resistant pathogens are responsible for 2 million illnesses, 23,000 premature deaths, and an estimated $20 billion dollars in health care costs per year (1,2). The rapid emergence of drug resistant pathogens threatens to undo nearly a century’s worth of biomedical advances, and the situation has become so dire that President Obama has recently made fighting antibiotic resistant pathogens a top national priority.

Engineering Bacteria

Figure 1. Azido modified KDO was used to metabolically glycoengineer the LPS core of E. Coli

Newly emerging molecular engineering techniques may lead the way for next generation therapies designed to attack resistant microbes. One such strategy is metabolic glycoengineering, which is using unnatural monosaccharides to intercept the metabolic machinery of a cell to artificially install chemical “handles” on the surface. These chemical handles can then be exploited by performing reactions known as “click chemistry” to connect almost anything a researcher can think of to the surface of any cell.

Some of the most important structures of bacteria such as the peptidoglycan layer, lipopolysaccharides (LPS), teichoic acids, and capsule are comprised of extensive amounts of carbohydrates. Using glycoengineering, a physician may one day be able engineer those structures with unnatural monosaccharides to disrupt the adhesive properties, directly image, or target drugs to bacteria in a species specific manner–an unprecedented level of selectivity currently unachievable with our current regimen of antibiotics (Fig. 1).

For further reading:
(1) http://www.cdc.gov/drugresistance/national-strategy/
(2) http://www.niaid.nih.gov/topics/antimicrobialResistance/understanding/Pages/quickFacts.aspx

About the Author: Christopher Saeui is a fourth year Biomedical Engineering PhD student in the Kevin J. Yarema Laboratory for Cell and Carbohydrate Engineering studying the epigenetic and metabolic mechanisms that alter glycosylation in cancer.

 

What is microfluidics?

Microfluidics continues to find applications in many fields as researchers are realizing the benefits of scaling down to micron scales. This has implications in saving money from reagents and time from completing lengthy assays.

It also means that researchers are able to control experimental parameters at the micron scale more effectively, and use the fluidic flow to provide a dynamic environment. Applications for these devices include, but are not limited to, examples such as pathogen and cancer detection from blood, forming microparticles, studying antibiotic drug-resistant bacteria, understanding nanoparticle blood transport, and observation of the kinetics of chemical reactions.

microfluidicdevice

Figure 1: A picture of a general microfluidic device. Source: http://blogs.nature.com/spoonful/2012/02/chip-promises-better-diagnosis-for-common-blood-disorder.html)

One reason that microfluidics has become so widespread is that the process to develop and create these devices is relatively simple and inexpensive. The process, called photolithography, is based off of a technology developed for the semiconductor industry in developing small features for circuits.

Photolithography uses special polymers that are reactive to certain wavelengths of light to create the forms used to make the device. Then another polymer, typically polydimethylsiloxane (PDMS), is poured into the casted photo-cured polymer mold to produce the microfluidic device. Many devices can be made from this mold and used in research and diagnostics for low-volume, high-throughput experiments.

John Hickey is a second year Biomedical Engineering PhD candidate in the Jon Schneck lab researching the use of different biomaterials for immunotherapies and microfluidics in identifying rare immune cells.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Chakravarthy: edging toward breakthroughs in nanomedicine

Krishnan Chakravarthy is a resident physician in the Department of Anesthesiology and Critical Care Medicine at the Johns Hopkins Hospital. He is also the founder of a company called NanoAxis. He is seeking potential faculty collaborators through Johns Hopkins Institute for NanoBioTechnology, where he is a recently affiliated faculty member.

Krishnan Chakravarthy

Krishnan Chakravarthy

Chakravarthy launched NanoAxis in 2009 while earning an MD and PhD at SUNY Buffalo. He won the Henry A. Panasci Jr. Technology Entrepreneurship Competition sponsored by the University of Buffalo School of Management with a business plan for creating a new drug delivery mode for seasonal and pandemic flu using quantum dots and gold nano particles.

He is the remaining founder and owner of NanoAxis and says the company has grown significantly since 2009. NanoAxis now has active labs and collaborators both nationally and internationally. The company is now taking root in Maryland, and Chakravarthy says he is actively working on collaborating with local biotech businesses in the area.

INBT: Can you briefly summarize your company’s main goals?

KC: At inception, we were primarily interested as a business to commercialize quantum dot technology for various industrial applications. Over the course of six years, our business goals have shifted from large-scale nano materials manufacturing to being the world leader in developing paradigm-shifting technologies in the field of nano medicine. For us, this comprises nano particle enabled advances in drug delivery and smart design point-of-care devices using nanotechnology.

At present, we have four main preclinical candidates that we are working on with the eventual goal of beginning FDA clinical trials. One project involves a novel antiviral therapy for seasonal and pandemic influenza that we are actively developing with the US Centers for Disease Control. This antiviral therapy also has shown to have therapeutic benefits in the treatment of Ebola virus.

In the realm of neuroscience, we have efforts to target Alzheimer’s disease, chronic pain, and depression using nano particles that deliver micro RNA and signal interference RNA designed to up-regulate and down-regulate key proteins. The preclinical data from both in vitro and in vivo studies look extremely promising.

Furthermore, we are currently in developing of two hand-held devices for screening for infections related to prosthetic implants (such as joint replacement). They could be used in the operating room and clinics across the US. Our goal is for orthopedic surgeons to be able to measure specific infection markers at their fingertips to enable them to make safe and cost-effective medical management decisions based on an accurate and precise screening tool. One of the devices will be iPhone compatible, while the other we are developing as a stand-alone device that would be ideal for markets in developing countries.

Our hope is that our platform will extend to diagnosing infectious diseases, be used in the Intensive Care Unit for basic metabolic panel measurements and be extended to a host of other medical applications. We will likely begin FDA clinical trials for these devices by early 2016.  I am also working on developing a platform for detection of various disease processes using nanotechnology and breath as a medium for detecting specific breath-based biomarkers.

INBT: What sort of collaborations are you hoping to establish?

KC: I am hoping to use the extensive knowledge at INBT and at Hopkins to help further our development efforts. As an affiliated faculty, I feel honored to be part of such an impressive think tank of scientists and entrepreneurs. I believe nanotechnology is at the heart of the bench-to-bedside paradigm. It is one component of the growing medical revolution that is happening around the world. In addition, unlike any other industry or science, nano medicine advances are going to be interdisciplinary and collaborative. So teamwork, collaboration, and collective ideas are going to push ongoing advances and development.

INBT: What are the main research challenges you would like to address?

KC: At present, the main stumbling block will be pushing our technology through FDA clinical trials. The process is long, tedious, and expensive. In addition, preclinical data has to be sound. So refinement of the nano materials to find the ideal candidate to deliver the gene or drug will require creativity and repeated experimentation. In addition, when thinking about the ultimate goal of seeing these drugs being used in patients, large-scale production becomes an important component to address.  It has to be of consistent quality, safe, and easily reproducible in large quantities.  So these are things we need to think about from an industrial perspective when you are no longer in the academic realm.

INBT: Anything else people should know?

KC: I am looking forward to working with other INBT faculty.  We are always open to new ideas, and my research team would be more than willing to start new projects.  Likewise, we will also try and suggest areas that we think specific faculty would be suitable for project development.

Visit the Chakravarthy Research Group Website: www.nanoaxisllc.com

Recent Publications:

Jacob A, Chakravarthy K (2014-04-06 14:52:34 UTC) Engineering Magnetic Nanoparticles for Thermo-Ablation and Drug Delivery in Neurological Cancers. Cureus 6(4): e170. doi:10.7759/cureus.170

Jacob A, Chakravarthy K, Law M, et al. (2014-04-21 18:33:15 UTC) Neuroradiology, Anesthesia, Bioengineering, and Hardware Programming in the Clinical Applications of Deep Brain Stimulation. Cureus 6(4): e172. doi:10.7759/cureus.172

Upcoming Invited Talks:

Designing smart nano-systems for effective gene and drug delivery across the blood brain barrier.  12th Annual World Brain Mapping and Therapeutics Congress.  March 6-8, 2015, Los Angeles, USA

Selective abrogation of IL-12/IL-23 production provides novel therapeutic modality in combating lethal synergism of influenza and secondary pneumonia.  5th World Congress on Cell Science and Stem cell research.  March 23-25, 2015, Chicago, USA

Periodically Johns Hopkins Institute for NanoBioTechnology (INBT) features a brief profile on one of its affiliated faculty members. If you are an affiliated faculty member of INBT and would like to be featured, contact INBT’s science writer, Mary Spiro at mspiro@jhu.edu. If you wish to become an affiliated faculty member visit this link. http://inbt.jhu.edu/apps/faculty/join/