It’s in the Genes: Advances in Genome-Editing Tools

by Esha Ghai

We have long been told that our DNA is unchangeable, often encouraging us to blame our flaws and accredit our talents to genetics. However, the introduction of genome-editing gives scientists the ability to alter genetic material and effectively change the basis of an organism. Genetic material can now be added, removed, or mutated at any specified location in an organism, allowing for cures to genetically inherited diseases and a wide range of illnesses. A recently discovered microbial ribonucleoprotein with such genome-editing capabilities is the CRISPR/Cas9 system. 

CRISPR/Cas9, short for “clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9”, was naturally developed by bacteria as a defense mechanism against invading viruses. As a part of this mechanism, bacteria confiscate fragments of DNA from invading viruses, and use the fragments to construct CRISPR arrays [1]. CRISPR arrays give the bacteria the ability to recognize identical or similar viruses, so if a similar virus attacks again, the bacteria produces RNA segments from the CRISPR array to specifically attack the virus’s DNA [1]. Cas9, an enzyme, is then used to cut apart the DNA, essentially killing the virus [1]. Scientists in the medical community are now adapting this gene-manipulation system to be used in cells in other organisms, including humans. The genome-editing tool has unlocked the potential to combat human genetic diseases previously deemed untreatable. A more cost-efficient, advanced, and precise method of gene-editing, CRISPR/Cas9 is revolutionizing the face of medicine as we know it [1].

Fascinated by the properties of CRISPR/Cas9 and microbes, Dr. Xue Gao, a professor of Chemical and Biomolecular Engineering at Rice University, currently investigates the applications of genome-editing and microbe-based natural product discovery. Her research “lies at the interface of chemical biology and biomolecular engineering with a primary focus on small- and macro-molecule discovery and their applications to human health, agriculture, and energy.” Dr. Gao’s contribution to the development of Statins, a pharmaceutical drug with lipid-lowering capabilities for those at risk of cardiovascular disease, piqued her interest in protein engineering. The frequent use of Statins in the medical society motivated Dr. Gao to continue synthesizing molecules to combat diseases. 

Her research also encompasses the use of enzyme-catalyzation (increase in the rate of a process due to an enzyme) for anticancer and antibiotic drug synthesis. Enzymes are powerful biological catalysts, accelerating chemical reactions by assisting in the formation of a transition state between the starting reagent and end product. Enzymes are also highly specific in which reactions it catalyzes, usually catalyzing only a single chemical reaction (or reactions very similar in nature), limiting side reactions and the ultimate formation of wasteful by-products [3]. According to Dr. Gao, whereas traditional chemical methods of synthesizing compounds require environmentally toxic solvents and harsh conditions, enzymes work well in moderate environments and are capable of producing a higher yield of 99%. As enzymes are proteins, extreme temperatures and pH levels result in denaturation, rendering the enzyme inactive. Therefore, enzyme catalyzation ability is enhanced at moderate conditions. In biological systems, a majority of processes utilize enzymes, from the transfer of CO2 in the body through the enzyme carbonic anhydrase, to breaking down sugars through the enzyme amylase. The development of enzymes to catalyze complex chemical reactions, called directed evolution, is a major focus of the Gao Laboratory. 

Dr. Gao ascribes “her love [for] exploring what has already been designed by nature” to the sheer intelligence of microbes and their methods of defense against viruses. “Microbes are super smart. They create protein encodings themselves to fight different types of environments.”  CRISPR/Cas9, another prominent example utilizing protein encodings and enzyme catalysis, has also attracted Dr. Gao’s interest. CRISPRs are specialized protein encodings, while Cas9 is an enzyme capable of cutting apart genetic material. Recently, Dr. Gao utilized the genome-editing capabilities of CRISPR/Cas9 to treat autosomal-dominant hearing loss. Autosomal-dominant diseases are inherited from an abnormal gene from one parent, even when the corresponding gene from the other parent is normal. Dr. Gao’s findings suggested that a targeted delivery of gene-disrupting agents such as CRISPR/Cas9 in vivo (within a living organism) can potentially treat such cases of hearing loss [2].

Dr. Gao’s research group is planning to do further work in CRISPR/Cas9 applications for a variety of human genetic diseases, and hopes to make breakthroughs in research through accommodating advanced computational tools and developing DNA sequencing technology (technology that determines the sequence of nucleotide bases). Dr. Gao envisions the next stage of her group’s research to be developing new enzyme-catalyzed reactions in the energy sector, particularly the production of biofuels, and combating food contamination in the agricultural industry. 

Dr. Gao’s current and future research aspirations in the field of human genetics and the energy sector serve as an inspiration for aspiring female scientists globally. She hopes that young females will follow their passion in STEM, and envisions a future where female scientists are not in the minority. Because of Dr. Gao and her vision, the Gao Laboratory is not only an embodiment of cutting-edge innovation in the future of enzyme-catalyzed reactions, but also the inspirational potential of female scientists. 

Works Cited

[1] What are genome editing and CRISPR-Cas9? - Genetics Home Reference - NIH. https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting (accessed Nov 8, 2019).

[2] Gao, X., Tao, Y., Lamas, V. et al. Nature. [Online] 2018, 553, 217-221. https://www.nature.com/articles/nature25164 (accessed Nov 8, 2019).
[3] Berg, J.; Tymoczco, John; etc. Biochemistry, 5th; W.H Freeman & Co: New York, 2002; 303-341.

Membrane Fusion And The Secret to Muscular Degeneration

by Lam Nguyen

In the human body, there are about 37 trillion cells. Each cell is made up of organelles that are busy creating proteins, digesting nutrients, and transporting different molecules or other organelles around. In order to facilitate the diverse functions different organelles assume, the plasma membrane, a barrier made up of proteins and lipids, helps compartmentalize different metabolic reactions to specific organelles and regions of the cell. Plasma membranes also play a critical role in the transportation of nutrients and signals between organelles through a process called fusion. A major topic within plasma membrane research, membrane fusion is a process in which two separate lipid membranes fuse into one single layer [1]. This process is essential in the creation of many organelles, such as the ER, a particular organelle that has fascinated Dr. James McNew, professor of biosciences, ever since he first came to Rice University.

Dr. McNew received his Bachelor’s of Science in Biochemistry from Texas A&M University, his Ph.D. in Pharmacology from UT Southwestern,  and he completed his postdoctoral fellowship at the Memorial Sloan-Kettering Cancer Center. His interest in cellular biology and membrane fusion started with his graduate work while researching how the peroxisome, an organelle responsible for various metabolic processes such as detoxification and cellular signaling, worked. This particular lab experience helped him realize that he had a passion for understanding basic molecular biology and “how different cellular components moved around the cell.” His burgeoning interest in basic cell biology followed him to his postdoctoral fellowship where his research centered around how proteins were moved, targeted, and carried to the Golgi apparatus, an organelle that processes and sends out newly synthesized molecules. Dr. McNew’s passion for cell biology and membrane fusion  has defined much of the work he has done here at Rice. His original research pertains to understanding membrane fusion in the endoplasmic reticulum (ER). The ER is a network of membranous tubules that is involved in protein and lipid synthesis1. Dr. McNew and Dr. Michael Stern, a fly geneticist and professor at Rice, observed how these tubules came together, but “the proteins involved were unclear.” They then hypothesized that the protein elastin, a protein present in connective tissue, played a significant role in ER membrane fusion. The two were able to confirm that elastin helps ER tubules come together, and they characterized the protein by studying it both in vivo (in a test tube) and back in fruit flies.

Dr. McNew also describes himself as a “basic cell biologist” and that his work is generally “far removed from something medicinal or therapeutic.” Work by clinicians on the elastin protein, however, has confirmed that elastin plays a role in physiological processes in humans. In fact, defects in the gene encoding for elastin play a role in hereditary splasic paraplegia (HSP), a group of genetic diseases where weakness and stiffness in the muscles gradually lead to muscular degeneration overtime.   This discovery has allowed Dr. McNew to connect his basic biology research to clinical applications. Alongside characterizing the mechanism of elastin in ER membrane fusion, Dr. McNew has also been deepening his understanding of how mutations in this protein can cause human diseases. 

Trying to understand how disruptions in the elastin and ER membrane fusion could cause the degeneration of muscles seen in HSP, Dr. McNew’s lab discovered that the loss of elastin function results in neurodegeneration which eventually leads to the deterioration of muscles. This explains how “people who have HSP lose motor control and their ability to walk.” Between adjacent neurons, small signaling molecules called neurotransmitters are transported  to send the electrical signal from one neuron to another. Dr. McNew observed that defects in elastin seen in HSP patients are somehow connected to the lack of neurotransmission. The lab was initially puzzled because they saw signs of muscle degeneration but not neurodegeneration. “Even though nerve damage is a known cause of muscle atrophy, conventional wisdom is that the nerve cells die first," Dr. McNew explains.  Consequently, Dr. McNew and his lab have begun investigating the cellular communication involved in this seeming paradox by learning about the other signalling molecules involved, specifically the torr kinase protein [2]. The McNew lab has been researching how the lack of neurotransmission is signalled by the torr kinase in order to provoke cell death in muscular tissue. 

Dr. McNew plans on building on the vast amount of data that his lab has obtained by further exploring other types of proteins that work with elastin and torr. Understanding these biochemical interactions will allow scientists to have a better picture of how elastin defects, lack of neurotransmission, and muscular degeneration are all connected to each other. The opportunity to discover new conclusions and to be on the forefront of scientific innovations is what often inspires undergraduates to get involved in research. When asked about if he has any advice for aspiring undergraduate researchers, he says that one has to be “okay with failure.” “It is very exciting and engaging to do something for the very first time. The feeling that you are the first person to do something is very addictive and keeps you going!” exclaims Dr. McNew. It is this excitement that will continue to inspire Dr. McNew in furthering his understanding of our cells and how they work.

Works Cited

(1) Daumke, O.; Praefcke, G. Structural Insights Into Membrane Fusion At The Endoplasmic Reticulum. Proceedings of the National Academy of Sciences 2011, 108 (6), 2175-2176.

(2) Loewith, R.; Hall, M. Target Of Rapamycin (TOR) In Nutrient Signaling And Growth Control. Genetics 2011, 189 (4), 1177-1201.

The Machines within Our Cells: Rewiring Genetic Circuits

by Gulnihal Tomur

Many people regard biology as simply trying to solve the mysteries of nature through observation.  A newly emerging field called synthetic biology bridges the gap between engineering and biosciences through reconstructing  already existing biological systems or fabricating entirely new ones by using artificial DNA synthesis technology. As synthetic biologists construct and insert manufactured genes in cells, they have two main goals in mind: either turn the cells into drug or biofuel factories or gain a better understanding of the roles different biological components play in naturally existing biological system. Synthetic biology allows scientists to reconstruct existing biological systems on a molecular level.

When asked to describe synthetic biology, Dr. Caleb Bashor of the BioScience Research Collaborative at Rice alludes to a famous quote from physicist Richard Feynman: “What I cannot create, I do not understand.” Dr. Bashor and his team attempt to rebuild intricate biological pathways to better comprehend their underlying principles. He refers to cells as micro-level machines — and “just like any machine, the cells have wiring inside them in the form of DNA and proteins.” Accordingly, Dr. Bashor’s approaches cellular systems from an engineering standpoint as he reconstructs these systems just like how an electrical engineer would rewire a machine to figure out how it works. “I am partly an engineer and partly a scientist, trying to understand how cells are organized so I can rewire them,” Dr. Bashor explained. 

Yet, Dr. Bashor did not start his scientific journey as a synthetic biologist; instead, he first occupied himself with biochemistry as an undergrad at Reed College. He was especially drawn to thinking about biology from the protein structure scale. At UCSF, he pursued the study of biological structures as a grad student from a perspective rooted in biophysics. Dr. Bashor got involved in synthetic biology at its early stages through a protein structure and function lab at UCSF, where they put different protein domains together to change the function of the protein as it was expressed in the cell. Dr. Bashor shares how they were essentially engaged in synthetic biology, yet did not know how broad the scope of their research was: “Eventually, people came along and told us ‘You guys are doing synthetic biology.’ And our reaction was ‘What’s that?’” As Dr. Bashor immersed himself more and more into this field, he dedicated his postdoc research at MIT to engineering transcriptional networks in eukaryotic cells. His current research at BioScience Research Collaborative encapsulates an even more ambitious approach as he and his team establish “synthetic regulatory circuits to reprogram the behavior of human cells.” Dr. Bashor and his team currently tackle mammalian regulatory circuits with potential applications that range from biosensing to cell-based therapy.

In order to fully understand Dr. Bashor’s work and its implications, we must first look into a natural gene circuit. Both eukaryotes and prokaryotes carry multiple genes in their genome, some of which get turned on and off depending on the external and internal signals cells receive. Since most proteins have complex structures, usually multiple genes are clustered together in a transcription unit. This then codes for the subunits of a single protein. The expression of these  genes are all dependent on a single promoter for their expression; the promoter serves as the site where RNA polymerase binds to DNA to initiate the transcription of the information embedded in DNA to mRNA. The so-called on-off switch of such transcription units is a specific DNA segment called an operator. Located either within or after the promoter, the operator is responsible for regulating when RNA polymerase can bind to DNA and start the transcription of the gene cluster. The promoter, the operator, and the clustered genes make up genetic circuits known as operons. 

When another protein called a repressor, which is the product of a regulatory gene separate from the operon’s gene cluster, is present in its functional state, it binds to the operator, thus physically blocking RNA polymerase from binding to the promoter. Internal and external factors, such as the presence of a specific molecule, influence the activity of the repressor by changing its 3D shape, which allows cells to switch on and off specific gene clusters based on environmental cues. Synthetic biologists take advantage of this knowledge as they introduce molecules in the cell which may turn on or off a repressor, add artificially designed circuitry into the existing systems, or isolate certain parts of a naturally occurring gene circuit through genetic modification. 

Mirroring and reconstructing such systems, however, still proves to be a real challenge despite the advancement in synthetic biology. One common problem scientists deal with is the instability of artificial gene circuits. These artificial circuits can break the natural balance of the metabolic systems of the cell. As the artificial gene circuits use the energy which would otherwise be used for growth and division, they hinder the cells from maximizing their fitness. Thus, any mutant cell in the cluster which did not accept the circuit outcompete the synthetically engineered cells. “The circuit becomes unstable evolutionary; the cells spit out the circuit over time in order to grow faster,” explains Dr. Bashor. Intrigued by this evolutionary process, he put his mind to engineering a circuit that doesn’t get spat out. He realized that he needed a device that would help him and his team monitor the evolutionary process of cells with artificial circuits in them. The result was eVOLVER — it enabled them to work with many circuits at a time. As an automated multi-culture platform, eVOLVER can control multiple parameters at once. It shows the cell growth and detects any disparities which may signal a mutant culture that spat out the circuit put into the system. “We can grow [the cells], watch the circuits break, and see where they recover their fitness as a result of the circuit breaking,” tells us Dr. Bashor. Ultimately, the device operates as a tool for being able to build circuits and then test their stability, which is a crucial factor if the circuit is desired to be stable for a long time so that, for instance, it can be used for the production of a chemical.

Another challenge scientists face presents itself in the complexity of multi-gene systems. Pleitropic genes, which influence two or more seemingly unrelated phenotypic traits, and gene clusters regulated by common factors hinder scientists who aim to understand the function of individual parts in such systems. Therefore, synthetic biologists turn to a technique called refactoring. “Refactoring is a term borrowed from computer science,” tells us Dr. Bashor, “somebody gives you a code that is really hard to work with, so you rewrite in a way that it is easier to change the parts individually and to see what you’re changing.” In a similar fashion, synthetic biologists “physically abstract genes away from their native genomic content.” (1) The new content in which scientists place the gene in question tends to be a simple plasmid. Not only does this new medium offer easier control over the effects and outcomes of the gene due to its simplicity, but it also allows scientists to quickly modify the new genomic content since putting a new piece of DNA in a plasmid is a relatively easy process. By eliminating the interference caused by the natural genomic context, synthetic biologists observe the role of specific genes in the whole system. Yet, Dr. Bashor warns us not to see interference as a sheer burden on the system. “You may make the regulation very simple, but then you lose something in the behavior. That’s a clue that the regulatory forces that are associated with that context are important.” In the end, he sees refactoring, and synthetic biology in general, as a trial-and-error process — synthetic biologists simplify and modify and rewire the systems they are curious about over and over again to master the role of individual parts, which then allow them to see the bigger picture more clearly.

There are many other obstacles in the synthetic biology scene scientists are still trying to overcome today. The inability to insert big chunks of DNA into cells, for example, is hindering scientists from taking full advantage of synthetic biology in cell therapy. Yet, Dr. Bashor sees the intersection of synthetic biology and cell therapy as one of the most promising developments to look out for in the future. As the trial-and-error process that is synthetic biology continues to ignite curiosity in current and future scientists, the field will offer more and more to the scientific community. From advancements in agriculture to biofuel production, the manipulation of biological systems has already enhanced our lives in ways we may not even realize, and it continues to help us finesse biological systems in ways we desire.

Sources

Synthetic Biology Explained. https://archive.bio.org/articles/synthetic-biology-explained (accessed Jan 20, 2020).

The Bashor Lab at Rice. http://bashorlab.rice.edu/index_large.html (accessed Jan 20, 2020).

The Solution Right In Front Of (or is it Inside?) Our Eyes

by Nithya Ramcharan

Antibiotics have for long been a prominent facet of medicine and are ubiquitous in households today due to their accessibility. At the earliest sign of a bacterial infection, we consume antibiotics with the confidence that it will be eradicated within a couple of weeks. However, this confidence has led to antibiotic abuse, a major issue in the medical field currently. For one, overusing antibiotics depletes the natural fauna populating certain organs like our intestines, making them more vulnerable to virulent bacterial strains. Naturally-occurring bacteria in our organs are a component of the immune system’s first line of defense and play a crucial role in immune system homeostasis and development [1]. Most importantly, antibiotic abuse has led to what some sources dub “the Antibiotic Resistance Crisis.” Antibiotics are often overprescribed when many of the infections’ pathogens are left unidentified, so out of caution, infections are assumed to be bacterial. 80% of antibiotics sold in the US are funneled into livestock for growth and prophylaxis [2].  As a result, bacteria are rapidly developing resistance to many commercial antibiotics and are returning more potent than ever. Bacterial genes can be transferred both to relatives and non-relatives through plasmids, circular DNA molecules in bacteria that can replicate independently of their host bacterial cell. This can confer resistance rapidly not only within a single population but also between different bacterial species [3]. Researchers are thus looking for alternatives to antibiotics to curb bacterial resistance and its catastrophic consequences.

One solution researchers have identified is a group of proteins occurring naturally within our bodies. Known as “antimicrobial peptides” (AMPs), these proteins are small, positively charged molecules that function within organisms as a natural immune response against microbes. Most commonly, AMPs can kill a bacterial cell through lysis; interactions between the positively charged clusters of an AMP and the negative charges in the phospholipid bilayer of a bacterium’s cell membrane form pores in the membrane, which ruptures the bacterium [4]. AMPs can also inhibit bacterial growth by targeting intracellular operations after permeating the membrane. They are present in organs in the human body most susceptible to infection such as the skin and the eyes [4]. Crucially, unlike antibiotics, bacteria have not developed solid resistance to AMPs, which is a key reason why AMPs offer a more attractive and effective method of treating bacterial infections. However, there is still little established research on how exactly AMPs neutralize bacteria. 

Professor Anatoly Kolomeisky of Rice University’s Department of Chemistry has been investigating the mechanisms of AMPs. His work started with the observation that antibiotic abuse can be attributed to people wanting to completely eliminate bacteria from their systems. His team developed a theoretical framework based on preexisting quantitative data to analyze how AMPs kill bacteria by considering bacterial growth as well as AMP entrance and inhibition rates into the bacteria as stochastic (randomly determined) processes. The model, in essence, consisted of a set of chemical reactions that would eventually lead to elimination of bacteria from the system. “We looked at the system as a sequence of states,” said Dr. Kolomeisky. “These states differed from each other only in the amount of AMPs.” In the occasion that the AMPs kill the bacterial cell, the corresponding state is removed. He called the model a “one-dimensional picture in a chemical space,” in the sense that it provided more of a black-or-white outlook: with certain factors, all the bacteria would either be killed or none of them would be killed. Although the model is simplified and every biophysical process is not accounted for, the model offers a convenient method for calculating the dynamics of the reaction, such as bacterial growth rate and bacterial clearance rate and the parameters that can modify these rates. Additionally, unlike other theoretical research on AMPs, this study takes into account the stochasticity of the processes of AMPs entering and inhibiting the cell [5]. This offers an effective means to further understand the mechanisms of AMPs that can be analyzed statistically and later compared with empirical data. 

Using the model, the team found out that the probability of inhibition and bacteria clearance rate have a positive relationship. Additionally, both processes of AMP entrance and inhibition of bacteria are equally important for AMPs to suppress bacterial growth. One finding is that higher heterogeneity in the AMPs facilitate faster entrance rates, while faster inhibition rates lead to lower heterogeneity among the types of AMPs. Controlling the heterogeneity in AMPs is key to balancing entrance and inhibition rates and thus achieving an optimal bacterial clearance rate [5]. Even though the stochastic model is simplified to fit the available information, the mean inhibition times predicted by the model are consistent with those observed in other studies [5]. 

Once these properties are understood, the applications of AMPs are numerous. They expand beyond inhibiting just bacteria and could suppress other pathogens. They can potentially be used in regulating cell division, facilitating wound healing, assisting in surgical healing, and suppressing cancer cells. AMPs activated by environmental factors such as pH have been developed for drug therapy. LL-37, an AMP found in humans, is one example of an AMP currently in commercial use. It has been identified to have bactericidal, anti-cancer, and anti-inflammatory properties [7]. Aside from medicine, they can be used in food production as natural food preservatives. One of them, pedocin PA-1 (produced by a diplococcus, a type of round-shaped bacterium), is used as a food preservative to prevent meat deterioration [6]. AMPs have also been identified as potential animal feed components to boost health and immunity in animal farming and aquaculture. AMPs with antifungal properties could be used as pesticides in the agriculture industry. 

However, much is left to discover about AMPs, as they are a diverse group of peptides that work differently depending on their environment; for example, some operate at the millimolar scale, while some require nanomolar concentrations to function. In addition, knowing how to optimize the entrance and killing rates is important in determining the ideal level of heterogeneity among the AMPs attacking the cell. Dr. Kolomeisky is advancing his current research on AMPs by investigating how using more than one type of AMP affects bacterial clearance. Implementing a “cocktail” mix, as he called it, of AMPs would kill bacteria more efficiently. Even though the rate of the reaction decreases as more types of AMPs are introduced, the probability of killing bacteria increases. He is also researching the dynamics of cancer development and cell cycle regulation, and could combine his research with AMPs to help better understand both processes. 

Once the mechanisms of AMPs are better known, they can revolutionize biomedicine. They can replace antibiotics in many medical treatments, thus mitigating the Antibiotic Resistance Crisis yet still allowing antibiotics to be used when needed. Reducing unnecessary usage of antibiotics will insulate them from the imminent threat of resistance developing in bacteria so rapidly. Beyond medicine, AMPs serve as better alternatives for inorganic chemicals used in food preservatives, growth promoters, and pesticides as they are naturally-occurring molecules that might be less likely to harm the organisms they aren’t programmed to target. In the nature of an archetypal journey of self-discovery, the solution to the antibiotic resistance crisis lay within us this whole time.

A Stressful Situation

by  Saksham Vashistha

Affecting nearly 80% of Americans, stress has become an integral part of our communities. However, while stress can affect our mental health, cells, too, experience physical stresses. However, while stress can affect our mood, physical stresses affect our bodies at the cellular level. Getting to the bottom of how cells respond to stresses is a useful tool in a wide variety of fields. Dr. Mike Gustin, a professor of biological sciences at Rice University, aims to understand stress responses in cells and their role in the treatment of cancer.

Dr. Michael Gustin’s passion for cellular biology developed during his time as a graduate student. While working in the Ching Kung lab at the University of Wisconsin-Madison, then-physiologist Dr. Gustin and his team were the first to discover that ion channels, special proteins that span the cellular membrane and help transport charged particles into and out of the cell, exist in yeast and bacteria. Now at Rice University, Dr. Gustin has made the transition to genetics, focusing on the impact of stress on cellular behaviors. His most recent works include collaborations with researchers across the Houston area, namely Dr. Erik Cressman, a chemist at MD Anderson. 

Dr. Gustin and Dr. Cressman worked together on an NIH funded project that investigates ways to treat liver cancer. Unfortunately, as Dr. Gustin points out, “for liver cancer, there is no good chemotherapy.” Oftentimes, cancerous tumors in the liver are inoperable and alternative treatments are required. However, Gustin and his team have found that “chemistry-driven exothermic reactions (heat-generating reactions) can ablate (remove) tissues in an increasingly controlled way.” There are a variety of different reactions that can be used to ablate cells. Dr. Gustin’s group decided to use a reaction between an acid and a base. By mixing the two solutions on the cell’s surface and allowing them to react, heat and a high solute concentration (osmolarity) are produced. This extreme heat combined with the high osmolarity is toxic to the cell and kills it.

As Dr. Gustin explains, “there is an ablation zone [on the cell’s surface] where the temperature is high enough to kill cells and there is a combination of not just heat, but also high osmolarity because of the salt that accumulates there.” Gustin’s group is interested in the peri-ablation zone, the area just around the region of high osmolarity.  It was hypothesized that the applied cellular stresses are followed by some types of cellular defense mechanisms or signaling pathways. These signaling pathways serve to protect the cell from physiological stresses, helping it survive the harsh ablation conditions. Understanding how these pathways work is key for ablation treatment in cancer patients. The goal is, as Dr. Gustin says, “to inhibit those pathways in a controlled way [to] control how much ablation actually happens.” Thus, by inhibiting certain cellular defense mechanisms, ablation in cancer cells can be modulated and used precisely during cancer treatment. 

One of the interesting finds during their investigation was that sometimes the stress applied to the cells prevented the cells from even responding. Upon further investigation, it was found that some of the genes that are activated for the stress response actually inhibited other stress responses. Dr. Gustin’s team attempted to understand these stress responses at the molecular level. By using computer modeling of known proteins that were involved in the stress response pathways, they were able to identify how the structures of the proteins were changed by the cellular stresses. Gustin’s team wanted to understand “what happens to the fluid around a protein and what happens when stresses combine around that protein.” Understanding what happens to the fluid around the protein can indicate what environmental changes the protein is experiencing. The alteration of the fluid condition can lead to altered protein structures. Changes in the molecular structure of proteins affects the cell’s ability to react properly to stress. This is because the structure of the protein is what determines its function. If the structure is altered in any way, the protein wouldn’t be able to carry out its function correctly. This molecular modeling approach can help scientists understand not only at the cellular but also the tissue and organ level of how stress responses operate in a mammalian body. Furthermore, ablation in tumor cells can be controlled via inhibition of the genes and subsequent proteins involved in the cellular stress response. This control over the cell’s genes can help make ablation more effective for liver cancer patients with inoperable tumors. 

Dr. Gustin’s investigation into harnessing the power of cellular stresses has the potential to change how physicians approach the treatment of cancer. By identifying mechanisms by which cancer cells resist ablation, Gustin’s work provides new insight into how physicians can directly alter cellular defense mechanisms to make them more vulnerable to treatment. While understanding the influences of stress on cells has made significant contributions in medicine, Gustin’s work can also have applications in other fields. Dr. Gustin has his eyes set on something greater. “I am really curious about … what happens during coral reef bleaching”, Dr. Gustin says with a glint in his eyes. Coral reef bleaching is a direct result of stresses (changing water temperatures) on the cells of the organism, causing them to lose the algae living in them. The algae are responsible for providing the majority of the coral’s energy, without them the coral starve and die.  Gustin hopes to use his knowledge of cellular stresses to “genetically engineer algae that stay in the coral” reagrdless of the water temperature in an effort to save an inumerable amount of coral around the world. Such an effort would help lead the effort to rebuild important coral reef ecosystems globally.

[1] Saad, L. Eight in 10 Americans Afflicted by Stress. https://news.gallup.com/poll/224336/eight-americans-afflicted-stress.aspx

[2] Fuentes, D.; Muñoz, N. M.; Guo, C.; Polak, U.; Minhaj, A. A.; Allen, W. J.; Gustin, M. C.; Cressman, E. N. K. A Molecular Dynamics Approach towards Evaluating Osmotic and Thermal Stress in the Extracellular Environment. International Journal of Hyperthermia2018, 35 (1), 559–567.

Can't Take MIONs Off You - The Future of Cancer Therapy

by Allison Wang

While Rice University may be famous for its part in the discovery of the buckyball and subsequent Nobel Prize in 1996, the research conducted by our university in the field of nanoparticles extends far beyond that. Roughly on the same size scale as proteins, nanoparticles range from 9 nm to 100 nm and have unique chemical, physical, and biological properties. They can be made from a variety of materials, such as semiconductors, polymers, and metals like gold and iron oxide. (1) Magnetic iron oxide nanoparticles (MIONs), are nanoparticles made out of iron oxides, usually magnetite (Fe3O4) or maghemite (γ-Fe2O3), and are nontoxic to the human body as they are broken down by the liver into iron and oxygen. (1) MIONs have an expanded range of applications for cancer therapy due to their superparamagnetism. When an external magnetic field is applied, the magnetic domains of the MION nanoparticles are synergistically aligned to produce a magnetization that is greater than the individual effects of each particle. (2) This induced magnetism is particularly useful for drug delivery, imaging, and tumor treatment. (1)

Professor Gang Bao, the Department Chair and Foyt Family Professor of Bioengineering and a Professor of Chemistry here at Rice, is the head of one such lab researching the applications of MIONs in different aspects of cancer treatment. He started out working with quantum dots around twenty years ago at the Georgia Institute of Technology and Emory University before being recruited to Rice through CPRIT in 2015. MIONs are able to enhance both the efficiency and specificity of drug delivery to a specific target tissue. The walls of our blood vessels are designed to only let small molecules cross from the bloodstream into the tissue, but by applying an external magnetic field to MIONs present in the circulatory system, the attachments between cells are disrupted. (3) This makes the blood vessel more permeable and allows drugs to extravasate out into bodily tissues. Additionally, by including nanoparticles into therapeutic cells or in the coating layer of drug particles, the applied magnetic field is able to direct the nanoparticle-facilitated treatment toward specific sites of the body. In fact, the nanoparticle surface’s applications in drug delivery can be further specialized to specifically target cancer cells. 

By altering the size, surface, and ligands of these nanoparticles, they can be adapted to target specific organs and cells for various applications. For one, the nanoparticles can be used during magnetic resonance imaging (MRI) as a contrast agent. MRIs are produced by applying a strong magnetic field to the body and detecting the spin polarization of hydrogen nuclei with a receiver. The contrasts seen in the MRI image is formed by the magnitude of polarization decaying at different rates in different tissues, which can be further changed through the interaction of the water protons with the nanoparticles. MIONs can also be coated with a radiotracer so that they show up better on fluorescence or PET scans of tumors. (2) By applying a magnetic field directly to a tumor, nanoparticles are attracted to that specific site and help create clearer images of the area. Similar to how nanoparticles can be loaded into drugs to aid with drug delivery, they can also be loaded into cell cultures to assist doctors tracking cells migrating through the body using MRI. (2)This is especially useful for monitoring stem cell treatments and their success. Another important use of nanoparticles lies in their ability to treat cancer through a therapy known as magnetic fluid hyperthermia (MFH), which utilizes the heating of iron oxide nanoparticles to kill or sensitize cancer cells. In MFH, a tumor is injected with nanoparticles and then exposed to an alternating magnetic field that causes the iron oxide to vibrate and generate just enough heat to destroy the cancer cells. (4) This sort of treatment is especially promising for pancreatic cancer; however, research is still ongoing on how to control the energy delivery and avoid non-specific heating of normal tissue.

Currently, the Bao lab is working on synthesizing iron oxide nanoparticles and gaining FDA approval for a small-scale clinical trial. As the field of nanoparticles is still relatively new, there are a lot of challenges that the lab faces before their research can be applied to therapeutic use in a clinic. First and foremost is producing enough particles for use. They currently are synthesizing milligram quantities of the nanoparticles, but the trials needed for FDA approval will require kilograms of nanoparticles. This cannot be achieved by simply scaling up the size of the machine’s reagents and reactions, as doing so would compromise nanoparticle quality. iLISATech, a company founded by Dr. Bao, is attempting to increase the production of iron-oxide nanoparticles to sufficient quantities for clinical trials, as well as for commercialization to sell to other laboratories and companies. Even after they are able to synthesize enough of the nanoparticles, there are still multiple phases and trials required by the FDA before a treatment can be approved for use by the public. As gold particle therapies have already been approved by the FDA, it is just a matter of more time and research before nanoparticle therapies will be approved and implemented in hospitals as a new treatment for cancer. 

Works Cited

[1] Wu, W.; et al. Nanoscale Res. Lett. 2008, 3, https://nanoscalereslett.springeropen.com/articles/10.1007/s11671-008-9174-9

[2] Landázuri, N.; et al. Small 2013 , 9(23), 4017-4026.

[3] Qiu, Y. et al. Nat. Commun. [Online] 2017, 8, https://www.nature.com/articles/ncomms15594

[4] Soetaert, F. et al. Adv. Drug Delivery Reviews 2020, 163-164, 65-83.

The Brains Behind Smart Wound Dressings

by Puneetha Goli

Did you know that nearly 1.35 million people die each year as a result of traffic crashes? Or that  injuries sustained from these accidents are the leading cause of death for people ages 5 to 29 (1)? Currently, those injured in accidents are required to wait for personnel to arrive and scope out the scene before any medical help from a wound care provider can be given. However, imagine if instead, in the time a person waits for emergency services, a “smart” band aid could be applied immediately to the wound, setting the wound until advanced medical follow-up is available. 

This is exactly what Dr. Swathi Balaji, an assistant professor of Surgery at Baylor College of Medicine, is working toward. Alongside Dr. Taiyun Chi, an assistant professor of electrical and computer engineering at Rice University, Dr. Balaji hopes to develop an artificial intelligence-assisted smart wound dressing to speed up the wound healing process. 

Dr. Balaji received a PhD in biomedical engineering from the University of Cincinnati after she fell in love with the applications of the field and recognized the impact she could make by combining her foundation in mechanical engineering with bioengineering.

“I came for a Masters in Mechanical Engineering at the University of Cincinnati,” said Dr. Balaji. “I loved the program, …[but] it became very obvious that we were [only] making small incremental changes.” Her Masters work served as a turning point in which she realized she wanted to enter a field where she could make a more long-term impact, leading her to pursue a PhD in bioengineering. “There was so much going on in terms of orthopedic research, biomaterials, regenerative tissue applications, so I was very fascinated with that,” said Balaji. 

The work that Dr. Balaji conducted as a PhD student on tissue engineering, as well as her subsequent work on understanding the regenerative mechanisms of fetuses has inspired her current projects at Baylor. The goals of her lab are to understand intracellular communications and how they translate into an individual’s response to injury, scarring, and wound healing. Through examining healing mechanisms in mouse models, Dr. Balaji has worked to establish a pipeline, ranging from basic investigations to more complex interactions with biomaterials to address her lab’s goals. 

Dr. Balaji’s interest in regenerative wound healing coupled with calls of action from the Defense Advanced Research Projects Agency (DARPA) to better understand what is occurring at the wound scene is what led to a collaboration between her and Dr. Chi and ultimately gave birth to the idea of this innovative smart wound dressing (2). 

Dr. Chi’s lab is working on the development of novel biology-microelectronics hybrid systems. The technology entails using advanced integrated circuits and microfabrication techniques - the same process of fabricating CPU, memory, and wireless transceivers chips in our cellphones and tablets (3) - to create miniature-sized electronic biosensors and bioactuators (4). Although the technology already exists, his sensors are unique in that they are one of the first that can record multiple biomarkers simultaneously and on a flexible membrane, ideal for human skin. Recognizing the potential interdisciplinary overlap, the two began discussing the possibility of integrating the research done in Dr.Balaji’s lab with Dr.Chi’s sensors in order to create a machine-learning system that would be capable of collecting information from a wound site and administering an individualized treatment. 

“It’s a very exciting project...,” said Dr.Balaji. “We started off with simple machine learning but the goal is to ultimately go into more advanced predictive modeling and make it smarter as we go on, and the dream would be ... to deploy this in the battlefield or when there is an emergency or when there is an accident - to be able to slap it on and let the wound healing happen right from the get-go.”

Currently, the project is in its initial stages. The teams have worked to perfect their respective independent technologies—Dr.Balaji’s team has developed their understanding of the science using animal models, while Dr.Chi’s team has refined the sensors and actuators. Now, they are currently working to integrate both of these components and apply them to a wound site. 

However, despite the preliminary nature of her work, Dr.Balaji already sees multiple avenues for the future of her research. One of her goals is to promote this niche field of science in an academic environment:  “I think our goal is to develop this new field of biology - machine learning microelectronics hybrid systems - either as a new course for undergraduate or graduate education or a new pipeline for promoting STEM-based education,” said Balaji. In addition to the educational angle, Dr. Balaji expressed an interest in getting the SMART wound dressing patented. In the future, she hopes for a world in which her SMART wound dressing could have a wide range of applications - from healing a scratch from a playground fall to something more complicated such as a burn wound. 

The potential of Dr.Balaji’s and Dr.Chi’s project has been recognized by the John S. Dunn Foundation Collaborative Research Award Program and the duo was awarded the 2020 John S. Dunn Collaborative Research Awards which was established to “fund projects with high potential for impacting human health”(5). “We were very excited..[and] we see a lot of potential,” Dr.Balaji said of winning the award. She believes the award will give her and Dr. Chi’s team the necessary resources to dedicate the time and effort to produce a final product that can be tested and examined. “I think at the current stage, we have all the different pieces of the puzzle, and over the course of the next two years, ...the Dunn Award is really going to help us bring our technology together. To come up with an idea that is received very well makes it a very, very rewarding experience.” 

References 

1. Road traffic injuries https://www.who.int/news-room/fact-sheets/detail/road-traffic-injuries (accessed Feb 24, 2021). 

2. Intelligent Healing for Complex Wounds https://www.darpa.mil/news-events/2019-02-06a (accessed Mar 3, 2021).

3. https://www.lifewire.com/what-is-cmos-2625826

4. https://www.nature.com/subjects/biosensors

5. Funding Opportunities – Gulf Coast Consortia https://www.gulfcoastconsortia.org/home/research/funding-opportunities/ (accessed Feb 24, 2021).

X Marks the Spot: High-Specificity Alternatives to Small Molecule Drugs

by Jessica Cao

“Side effects may include headache, dizziness, nausea, and vomiting, and serious, sometimes fatal allergic reactions may occur.” Most common and modern-day pharmaceuticals, such as those seen in medical advertisements, are classified as small molecule drugs (SMDs). These are defined as drugs with low molecular weight and are “capable of modulating biochemical processes to diagnose, treat, or prevent diseases”. [1] The nature of SMDs makes them particularly attractive in the pharmaceutical industry, as they can be easily manufactured and regulated compared to more complex compounds. This ease and convenience in turn lowers costs, allowing them to be more accessible and affordable for patients. [2] Unfortunately, the simple composition of SMDs typically results in a litany of unwanted side effects due to their inability to target specific protein receptors.The physiological dangers of these side effects have spurred the scientific community to discover other, potentially safer methods of drug delivery. 

Among these researchers is Dr. Jerzy Szablowski, an Assistant Professor of Bioengineering at Rice University. Dr. Szablowski’s research focuses on neurotherapeutics, particularly the development of technologies that can noninvasively monitor and interact with the brain.3 One of the projects at the center of his research is gene therapy. Dr. Szablowski believes that this is a distinctly promising method of drug delivery “because it can be separated into different layers of specificity.” He states that, unlike small molecule drugs, where “everything is encoded within several atoms of the molecule”, gene therapy can be optimized for specificity and modularity. Gene therapy involves the delivery of a new gene to a cell through a viral vector and a promoter, which is a DNA sequence that helps facilitate the production of the protein encoded by the gene. This type of multifaceted system is advantageous because each component can be modified for different uses. Since the purpose of the viral vector is to deliver the gene (the therapeutic component), the vector can be modified to treat specific parts of the body without altering the treatment. Furthermore, Dr. Szablowski elaborates that “the promoter [of the gene] determines subtype, [which can] provide specificity.” Ultimately, the ability to modify each  component of gene therapy without affecting the others yields greater specificity for each drug. Greater specificity allows for the avoidance of side effects resulting from non-specific binding between the drug and body receptors that are unrelated to the disease being addressed. However, assessing neurological illnesses to determine necessary treatment is often difficult, as brain biopsies are highly invasive and risky. 

In order to gain a better understanding of the pathology of neurological disorders and the effects that they have on specific parts of the brain, Dr. Szablowski and his lab have employed a method of controlling and monitoring brain tissue via ultrasound. This noninvasive technique involves focusing ultrasounds on a specific part of the skull to open the blood-brain barrier (BBB) by a few millimeters. As Dr. Szablowski describes, the ultrasounds are delivered in conjunction with “a special contrast agent that is FDA-approved, [which is] basically a bubble that is encased in a lipid. Because this contrast agent has gas, it can actually become compressed or [expanded].” He further specifies that ultrasounds are essentially “an expansion and compression of a mechanical wave over time”, which pushes apart blood vessels in the brain, causing the [impermeable] junctions of the BBB to separate, and the endothelium (a thin membrane) in the brain to become more permeable. During this stage, a patient’s blood can be drawn, and a simple blood test can be used to assess their neurological condition.4 This procedure can alternatively be used to deliver therapeutic nanoparticles and proteins such as antibodies to specific parts of the brain. Although the concept of opening the BBB may sound risky, Dr. Szablowski assures that “several hours post-application, the impermeable junctions basically close up, and they become normal again.” Furthermore, little to no side effects have been observed apart from minor brain inflammation, which is easily treated with the steroid dexamethasone. Dr. Szablowski believes that this method of focused ultrasound can be used independently or in conjunction with gene therapy to ultimately become a common method of highly specific drug delivery to the brain. 

Dr. Szablowski also hopes to expand on his current projects to discover novel methods of low-cost drug delivery, such as non-genetic therapy. He mentions that non-genetic therapy involves the “delivery of proteins that actually produce the drug on site, and the production of the drug on site allows it to be specific.” In other words, the biological site(s) involved with a disease are the only sites being treated, with other parts of the body remaining unaffected. Furthermore, the Szablowski lab has recently begun the Therapeutic Improvement Project, which will potentially simplify the arduous and expensive process of drug development. Dr. Szablowki says that currently, when developing a drug, “you first have to make a cell model of the drug, [then] find what receptors are responsible for the disease, then test [using a] tissue culture and mouse or larger animal, and if you're lucky and pass through all those stages, you can [use on] humans.” Unfortunately, because animals and humans are biologically different, a drug can affect each organism differently, and projects often remain “stuck” at the animal model phase. Dr. Szablowski and his lab are hoping to bypass the many obstacles of drug development by creating “a single drug [that] can treat multiple diseases by just delivering to single cells.” 

Ultimately, Dr. Szablowski hopes that his research will be applied to a number of diseases—most notably neurological ones such as Parkinson’s, epilepsy, and PTSD—as opposed to a specific illness. Many of these diseases are currently difficult to assess, monitor, and treat in real time, but Dr. Szablowski believes that these minimally invasive, yet highly specific methods will eventually allow the development of therapeutic treatment with little to no side effects. 

[1] Ngo, H. X.; Garneau-Tsodikova, S. What are the drugs of the future? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072476/ (accessed Feb 24, 2021). 

[2] Gurevich, E. V.; Gurevich, V. V. Therapeutic potential of small molecules and engineered proteins. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513659/ (accessed Feb 24, 2021). 

[3] Szablowski, J. Jerzy Szablowski: The People of Rice: Rice University. https://profiles.rice.edu/faculty/jerzy-szablowski (accessed Feb 24, 2021). 

[4] Miller, B. Targeting ultrasound for noninvasive diagnosis of brain cancer: The Source: Washington University in St. Louis. https://source.wustl.edu/2020/08/targeting-ultrasound-for-noninvasive-diagnosis-of-brain-cancer/ (accessed Feb 24, 2021).

The Mighty Might-ochondria

by Pavithr Goli

Famously known as the “Powerhouse of the Cell”, the mitochondria is vital as it provides the human body with the necessary energy to function on a day-to-day basis. In addition to this main role, researchers have recently discovered other mitochondrial functions critical to human survival such as interacting with pathogens during infections and metabolizing cholesterol (1). These often overlooked features of mitochondrial function have intrigued Dr. Natasha Kirienko, an Associate Professor in the Department of Biosciences at Rice, who has been studying mitochondrial surveillance pathways and damaged mitochondria. Furthermore, Dr. Kirienko has spent time studying how both can be utilized in anticancer therapeutics, which can potentially change the approach taken to treat cancer, saving numerous lives in the process.

Dr. Kirienko joined Rice University in 2015 after she earned a $2 million grant from CPRIT (Cancer Prevention & Research Institute of Texas). After earning her undergraduate and graduate degrees in biochemistry and biology in Russia, Dr. Kirienko came to the United States to work on her Ph.D. After receiving her Ph.D. in molecular biology from the University of Wyoming, Dr. Kirienko worked as a postdoctoral research fellow at the Massachusetts General Hospital and Harvard Medical School before joining Rice to continue her groundbreaking research on mitochondria. Dr. Kirienko developed her fascination for mitochondria during her time in graduate school where she was interested in why “mitochondria had lower efficiency in leukemia” and why “they had a poor ability to convert electrons into ATP.” 

As someone who has “always [been] interested in identifying fundamental principles of why things are working,” Dr. Kirienko was curious about the effect of disease on mitochondria and, “the biochemical basis of the cancer cells /cell lines.” This interest in studying the pathogenesis of mitochondria propelled her to begin conducting research and investigating the response of damaged mitochondria and the strategies these damaged structures employ to recover when pathogens impact their function. 

Dr. Kirienko spent years dedicated to developing a greater understanding of mitochondria with the hope that a new therapeutic method to fight cancer could be developed. This interest in understanding fundamental principles led Dr. Kirienko to focus her research on one of the most fundamental and basic organisms that exist: Caenorhabditis elegans or C. elegans (2). A roundworm commonly used in scientific research as a model organism, C. elegans has been instrumental in understanding the various molecular and genetic mechanisms the human body employs when responding to diseases like cancer (2). 

Using this model organism and previous literature as a guide, Dr. Kirienko has been able to observe novel pathways in mitochondria and find drugs that induce mitochondrial damage, pushing “cancer cells [to] undergo cell death like paraptosis or apoptosis.” The goal of Dr. Kirienko’s current approach is to apply this novel method of inducing cell death on ''different cancer cell types” or combine it with current anticancer drugs in a synergistic manner to create highly selective treatments capable of killing cancer cells and sparing healthy ones. This approach will help reduce the side effects commonly seen in cancer treatment while maintaining the treatment’s effectiveness.

To further her research, Dr. Kirienko uses an interdisciplinary approach by working with physical chemists and doctors to help ensure that her novel cancer therapy will be applicable beyond C. elegans. She hopes that physical chemists can optimize the drugs that Dr. Kirienko found by “synthesizing and improving the physical structure of the drug.” After further testing on mice and cell cultures, Dr. Kirienko intends to expand her research and consult the help of physicians in a potential clinical study.

Along with her cancer research, Dr. Kirienko has expanded her lab’s focus on other diseases caused by poor mitochondrial health such as Alzheimer’s disease. Specifically, she and her lab are trying to understand whether improving mitochondrial health may be beneficial for neuronal function. In order to understand this mechanism further Dr. Kirienko is attempting to use various biomolecules to avoid cellular death by activating mitochondria and “[finding] a way to help mitochondria fix themselves quicker.” 

Furthermore, Dr. Kirienko and her lab are attempting to observe mitochondrial reactions to infections. Using C. elegans as their model organisms, they are interested in “looking at basic toxins that damage mitochondria.” One specific group of toxins that they are investigating are siderophores, which are “small molecules with a peptide side chain… [that] are secreted in the body and release iron.” During a bacterial infection, the bacterial pathogen uses its siderophore to absorb iron from the host, enabling the bacteria to grow and infect the cells. The mitochondria are the site of electron transport chains (ETCs), which contain several iron-rich proteins that help generate energy for the cell; unfortunately, when “iron is stolen, the mitochondria get damaged.” (3).To better study this mysterious process, Dr. Kirienko and her lab have “developed a cell culture model where [they] can assess the pathogenicity of bacteria.” They aim to use this newly developed model to elucidate the reactions and mechanisms that allow pathogenic bacteria to hijack cellular mechanisms.

Beyond discovering mitochondrial pathways and reactions to infections, Dr. Kirienko's lab is eager to explore "mitophagy or mitochondrial turnover, applied to cancer… [and] study more mitochondrial pathways.” She hypothesizes that this research can provide key information in finding ways to strengthen mitochondrial health so that mitochondrial cells can effectively withstand deadly pathogens and diseases, ultimately leading to longevity in human health. 

Dr. Kirienko and her lab have been working on some groundbreaking research for the past five years. Using novel methods and ingenuity, Dr. Kirienko has already made major strides in the field of cancer therapy and molecular biology. Through her current projects and the future endeavors that she hopes her lab can make, Dr. Kirienko intends to understand more about the powerhouse of the cell and use it to develop influential cancer therapies. 

Works Cited:

(1)Wallace, Douglas C. “Mitochondria and cancer.” Nature reviews. Cancer vol. 12,10 (2012): 685-98. doi:10.1038/nrc3365  

(2)Why Study C. Elegans?, Dr. Lundquist, 01/19/21, www.people.ku.edu/~erikl/Lundquist_Lab/Why_study_C._elegans.html

(3) Montine TJ, Morrow JD. Fatty acid oxidation in the pathogenesis of Alzheimer's disease. Am J Pathol. 2005 May;166(5):1283-9. doi: 10.1016/S0002-9440(10)62347-4. PMID: 15855630; PMCID: PMC1606384.

A Memorable Discovery: Tracking Alzheimer’s with Light

by Jessica Cao

Every 65 seconds, someone in the United States develops Alzheimer’s disease [1]. Alzheimer’s disease is the most common form of dementia, a general disease marked by a decline in cognitive function severe enough to interfere with daily function. Alzheimer’s in particular is characterized by cognitive impairment and memory loss, and is most prevalent in people ages 65 and older [2]. These symptoms associated with the disease appear to be similar to aging, but are not part of the natural aging process [2]. There is currently no cure for Alzheimer’s, with current drugs only treating the symptoms (memory decline, confusion and disorientation), but significant developments have been made in the past few years towards understanding the disease. Among those in the research community advancing Alzheimer’s research is Dr. Angel Martí, an Associate Professor of Chemistry, Bioengineering, and Materials Science and NanoEngineering at Rice University. Dr. Martí’s lab is focused on the treatment and diagnosis of amyloid-related diseases such as Alzheimer’s and Parkinson’s disease, both of which are centered around the Beta Amyloid Hypothesis. 

The exact cause of Alzheimer’s is unknown, but several hypotheses have been formed in the past decades that may explain how the disease develops, most notably the Beta Amyloid Hypothesis. Beta amyloid (Aβ) is a sticky, hydrophobic protein found naturally in the brain in the form of monomers, its smallest molecular form. The monomers are derived from a larger protein known as Amyloid Precursor Protein (APP), whose original function is unknown. In patients with Alzheimer’s, Aβ monomers begin to aggregate and form Aβ oligomers; these oligomers may then aggregate further to form Aβ plaques, the most pathogenic form of Aβ [3]. Although Aβ plaques have been the main focus of Alzheimer’s research, Dr. Martí believes that understanding the development of Aβ oligomers—the aggregated form of Aβ proteins—is equally critical. Unlike plaques, Aβ oligomers are soluble in the blood and tissue fluid, allowing them to diffuse to different parts of the brain. This is particularly damaging since the oligomeric form of Aβ kills neurons as it diffuses. As Dr. Marti describes, “if Aβ is toxic [and] kills neurons, then as neurons start to die in patients with Alzheimer’s, the symptoms associated with [Alzheimer’s disease] will start.”

Taking this into consideration, Dr. Martí and his lab have created a ruthenium-based fluorescent complex, based on a concept known as fluorescence anisotropy, which tracks the development of Aβ oligomers in the brain. Fluorescence anisotropy is centered around the polarization of light, where “molecules can only absorb light that is in the direction of their transition dipole moments”. In other words, molecules can only absorb polarized light if they are oriented in the right direction, and will then rotate and emit light, resulting in a different polarization. However, since the molecules are extremely small, their high rotation speed results in the emission of light in multiple directions, depolarizing the light; the molecules can only emit polarized light in a specific direction when rotated more slowly. “Using fluorescence anisotropy, what you can measure is something that is proportional to how fast the molecules can rotate in solution.” The speed of the molecules is measured using a probe, which can bind to molecules if they are large enough. Aβ monomers are very small and are unable to interact with the ruthenium complex, “but once Aβ monomers start coalescing into bigger oligomers, then the probe can bind to the oligomers. And then, that probe that was rotating very, very fast before now rotates at the same rate as the big oligomer.” Using this complex, Dr. Martí and his lab may eventually be able to track the development of Aβ monomers into oligomers in Alzheimer’s patients by monitoring the changes in the speed that the probe rotates.

What can this be used for? The ruthenium-based fluorescent complex is still being refined and developed, but Dr. Martí says that it will eventually be used to “visualize or track [Aβ aggregation] in real time,” which will allow his lab to “test different drugs or molecules that might interact with these oligomers, and either break them apart or inhibit their formation”. Beyond its various applications, Dr. Martí believes that fluorescent complex may be further improved or by modifying the metal complex for increased binding efficiency, and possibly even modifying it to bind to proteins of other diseases. While this complex is not a cure for Alzheimer’s, it is undoubtedly a significant development in tracking the impact of potential cures. 

Works Cited

[1] Alzheimer’s Association. Alzheimer’s Disease Facts and Figures. https://www.alz.org/alzheimers-dementia/facts-figures (accessed Nov. 15, 2019).

[2] NIH National Institute on Aging. What is Alzheimer’s Disease? https://www.nia.nih.gov/health/what-alzheimers-disease (accessed Nov. 15, 2019). 

[3] Alzheimer’s Association. Beta-amyloid and the Amyloid Hypothesis. https://www.alz.org/national/documents/topicsheet_betaamyloid.pdf (accessed Nov. 15, 2019).

[4] Rice University. Angel Martí Group at Rice University. https://martigroup.rice.edu/(accessed Nov. 15, 2019).

One Mouse at a Time: Answering the Alzheimer’s Conundrum

by Mabel Tang

Alzheimer’s disease is the sixth leading cause of death in the United States [1]. One third of American seniors die with Alzheimer’s or dementia, and 5.8 million Americans live with the disease [2]. This number is projected to reach 14 million by the year 2050 [3]. Despite the large numbers of people affected by the disease, it has no cure. 

What we do know, however, is that there are several causes of Alzheimer’s disease. Studies suggest that the accumulation of proteins such as alpha-beta peptides and the tau protein in the brain contribute to the development of Alzheimer’s [4]. These aggregated proteins most likely disrupt neural activity and other circuits that control and support learning and memory, resulting in the death of neurons. Alzheimer’s can also be hereditary, although deterministic genes, or genes that directly cause disease, make up a very small percentage of cases [5]. On the other hand, Alzheimer’s risk is increased through head injury, diabetes and obesity, and age [6]. 

Dr. Joanna Jankowsky, a professor of neuroscience, neurology, neurosurgery, and molecular and cellular biology at Baylor College of Medicine, is seeking to better understand this devastating disease by modeling it with specially engineered mice. When Dr. Jankowsky first started her Ph.D at the California Institute of Technology, she was studying models of learning and memory. However, after working with a neurosurgeon on models of epilepsy, she found what she considers “the ground truth of the human disorder... You have a whole population of patients who want research on their disease because it’s so traumatizing for their life and their families’ lives. [My research] became much more grounded in something that seemed worthwhile. I really liked learning and memory, but I loved doing things that were clinically relevant, and the easiest way was to study a disease of learning and memory, which is how I came to Alzheimer’s disease.” 

Dr. Jankowsky’s lab seeks to understand age as a risk factor for Alzheimer’s, the genetics behind Alzheimer’s resilience, and the connection between Alzheimer’s pathology and resilience to the disease. According to Dr. Jankowsky, some patients who died cognitively healthy are found at autopsy with brains that should have caused dementia in their lifetime. To answer these questions, the lab uses mouse models, putting genes in and taking genes out of their genome to better emulate the conditions the lab wants to study. By engineering the mouse genome such that the mouse will develop Alzheimer’s-like pathology or taking out genes responsible for cognitive resilience to the disease, Dr. Jankowsky and her lab can see how the interactions of brain cell proteins affect the mouse’s cognitive behavior, and decipher if these alterations contribute to the development of Alzheimer’s. 

Dr. Jankowsky’s lab has two electrophysiology rigs, used to record the electrical activity of single neurons or circuits in the acute brain slices of their genetically engineered mice. According to Dr. Jankowsky, it is used to specifically look at how adding a gene or taking a gene away can change a neuron’s function. Her lab also uses stereotaxic surgery, a form of surgery used to inject viruses that specifically inject a protein of interest to a specific cell population or circuit in the brain. Finally, there are special behavioral rooms used to test cognitive function in mice and examine if the gene manipulations are impacting the mice’s ability to learn and retain new information.

Dr. Jankowsky’s research is supplemented by the developments in Alzheimer’s research in recent years. Scientists have found an association between a buildup of plaques of the amyloid-beta protein in between neurons and abnormal accumulations of the tau protein in neurofibrillary tangles with the development of Alzheimer’s disease. The accumulation of beta-amyloid in between neurons disrupts cell function, while neurofibrillary tangles block neuronal communication in areas that control learning and memory [7]. According to Dr. Jankowsky, her lab has been able to treat this amyloid pathology in mice with signs of Alzheimer’s disease and rescue the mice’s cognitive impairment by turning genes off and on. Most recently, her lab has been working to study particular circuits that are affected early in Alzheimer’s development, such as the circuit responsible for ongoing recall of familiar environments. “One of the problems Alzheimer’s patients will have, in the beginning of the disease, is that they will often end up in places that are familiar, but they suddenly realize they don’t know how to navigate out of that place,” Dr. Jankowksy says. “ We’ve been able to show that one of the circuits that is affected early in Alzheimer’s disease may underlay that deficit.” The finding Dr. Jankowsky believes is most relevant to human applications, though, is a study she completed in which she combined two ways of attacking the amyloid-beta protein. “We got a better effect on cognitive recovery than we did with either [treatment] alone, show[ing] that combination treatment may be more effective than single treatment. My hope is that it will be soon be conveyed in human studies, where so far we have done one drug at a time and failed every time. I hope we get to a point where we start considering combination trials in human studies too.”

These developments in research could not have occurred without technological improvements from just the last ten years. “Our access to knowledge [and] our ability to do large experiments quickly has increased exponentially,” Dr. Jankowsky recalls. “Things have progressed tremendously even in just the last ten years.” Despite this progress, however, the lab still faces challenges. According to Dr. Jankowsky, funding is always a challenge. The process of applying for grants is tedious, and there is a possibility of waiting a very long time between the time of application and when funding is actually received. While the funding rates for Alzheimer’s research has increased significantly in the last twenty years, Dr. Jankowsky says that “[scientists are] constantly struggling to juggle grants and employment”. Aside from funding, Dr. Jankowsky says that there are certain fundamental questions that still have not been answered. She is using her $450,000 Zenith Fellows research grant from the Alzheimer’s Association to work on a particular protein called T-mem106B. This membrane resides in the cell’s lysosome, which is considered the “trash bin” of the cell, degrading and recycling proteins to be used by the cell. However, it is not known what T-mem106B does at the lysosome. The gene that encodes this protein was found in subjects who were cognitively normal at death but had a lot of amyloid plaques and neurofibrillary tangles in their brains. The lysosome is supposed to dispose of these aggregates, and, as Dr. Jankowsky explains, “if our gene is responsible for making the lysosome work better, those patients should have been cognitively resilient because the lysosome was working better and got rid of all those protein aggregates. But it’s just the opposite.”

Despite these obstacles, Dr. Jankowsky hopes to see a brighter future for Alzheimer’s research. While she considers her laboratory a “very small cog in a very large wheel of research” and a “small part of what needs to happen in order to impact society,” she says the ideal goal is an interventional treatment and a cure for the disease. However, she would also settle for preventative treatments. “We are telling people to go out and live healthier lives. Those are all true, but there are a lot of people midlife like me who aren’t going to exercise as much as we should or don’t eat or sleep properly and are putting ourselves at risk. I hope there is something out there for those of us who aren’t doing everything we can to protect our brain health later in life.” Out of her work, however, Dr. Jankowsky considers what she can do best toward the larger effort of Alzheimer’s research is training the next generation of scientists. “If the people who come out of my lab learn how to do research in a rigorous way and become better scientists than I am, then I would have succeeded. I think I have a greater impact on the future with the people I train than I do with the publications I put out now.” 

References

[1] Alzheimer’s Association. Facts and Figures. https://www.alz.org/alzheimers-dementia/facts-figures (accessed Oct 22, 2019).

[2] Alzheimer’s Association. Facts and Figures. https://www.alz.org/alzheimers-dementia/facts-figures (accessed Oct 22, 2019).

[3] Alzheimer’s Association. Facts and Figures. https://www.alz.org/alzheimers-dementia/facts-figures (accessed Oct 22, 2019).

[4] Mucke, L. Alzheimer’s disease. Nature [Online] 2009, 461, 895-897. https://www.nature.com/articles/461895a#citeas (accessed Feb 20, 2020).

[5] Mucke, L. Alzheimer’s disease. Nature [Online] 2009, 461, 895-897. https://www.nature.com/articles/461895a#citeas (accessed Feb 20, 2020).

[6] Mucke, L. Alzheimer’s disease. Nature [Online] 2009, 461, 895-897. https://www.nature.com/articles/461895a#citeas (accessed Feb 20, 2020).

[7] Brion, JP. Neurofibrillary tangles and Alzheimer’s disease. Eur Neurol [Online] Oct 1998, 130-140. PubMed.gov. https://www.ncbi.nlm.nih.gov/pubmed/9748670 (accessed Oct 22, 2019).

ATAC: A Brain Therapy of the Future?

by Sophianne Loh

Rice University students and faculty are said to possess “unconventional wisdom.” We aren’t afraid to dream of solutions that seem impossible, push past limited frameworks of thought, and leap into uncharted territory. This ideal is perhaps most apparent in the neuroengineering research done at Rice. For years, neurological conditions like Alzheimer’s or epilepsy have been treated in much the same way: surgical procedures, injections, and electrical stimulation of the brain. [1] These treatments aim to “fix” dysfunctional neural circuits, which are often the origin of these neurological and psychological disorders. Unfortunately, not only are these options invasive, but they fail to target specific dysfunctional neurons, leading to adverse effects on the surrounding healthy brain tissues. 

As a result, researchers around the world have been hard at work creating smaller implants and less invasive electrodes on the scales of millimeters or even micrometers. [2] For example, researchers at the Korea Advanced Institute of Science and Technology recently designed a quarter-sized wireless implant that successfully modulated mouse brain neurons. [3] However, continuing to travel down the same path of surgical implants eventually leads to the same major issues: unwanted invasiveness, limited specificity of targets, and, most prominently, obtaining sufficient funding. As Dr. Jerzy Szablowski of the Rice Bioengineering Department points out, “If you look at the amount of funding we have for brain disorders - for example, from National Institutes of Health - you'll find out that the amount of money we spend on research that aims to treat brain disorder is very low compared to the burden that they cause” In fact, Dr. Szablowski notes the cost of creating one new drug for each brain disorder can be up to $1 billion. In comparison, the National Institute of Mental Health dedicated only $2.1 billion in 2021 to research on brain disorder treatments. [4] With over 1,000 neurological disorders affecting over 100 million individuals in America, there is an ever-growing need for less costly and more versatile treatments. [5]

In true Rice fashion, Dr. Szablowski approached this need by traveling down a different route. He wanted to create a one-stop, noninvasive, affordable treatment for multiple neurological disorders. In other words, what if a single technology could treat a variety of  neurological disorders with elegant simplicity? Dr. Szablowski has found an answer in acoustically targeted chemogenetics, or ATAC. Developed by Szablowski and advanced by his researchers in the Laboratory for Noninvasive Neuroengineering, ATAC is the first-ever fully noninvasive technique to control neurons with spatial, cell-type, and temporal precision. When applied to the treatment of neurological disorders, ATAC could treat a variety of disorders, as long as they rely on abnormal neuronal activity.

Despite the simplicity of the resulting treatment path, ATAC is a complex, multi-step process. It starts with the blood-brain barrier (BBB), a collection of different types of cells tightly joined together that line the blood vessels of the brain. The BBB serves to block certain pathogens or other harmful molecules in the bloodstream from entering the brain. However, this also means that the BBB won’t normally allow the passage of therapeutic molecules into target areas in the brain. [6] This is where the Szablowski lab turned to FUS-BBBO, or focused ultrasound blood-brain barrier opening, in their development of the ATAC method.

FUS-BBBO is a technique that had been well-researched prior to Dr. Szablowski’s development of ATAC. This ability to build upon a foundation of past research while pioneering a new treatment was important to Dr. Szablowski. He explains, “I wanted to find a method that is well-validated and something that would be safe for human use, and the fastest way to do that is to just look at what is available.” FUS-BBBO (Fig. 1) first involves the injection of microbubbles (typically a lipid or protein shell containing perfluoropropane or sulfur hexafluoride gas) into the desired blood vessels. Immediately after, short, low-pressure ultrasound wave pulses are applied to the BBB target site. This causes the microbubbles in the blood vessels to oscillate, a phenomenon termed stable cavitation. The oscillations apply pressure to the surrounding fluid, which opens the tight junctions between the cells of the BBB temporarily and reversibly. As a result, therapeutic molecules in the bloodstream can go past the BBB to reach neurons in the brain. [7]

Figure 1. (A) Schematic depiction of FUS-BBBO procedure. (B) MRI-guided FUS-BBBO procedure. [6]

Where Dr. Szablowski departs completely from previously known procedures is by combining FUS-BBBO with AAVs, or adeno-associated viruses, that encode engineered protein receptors that can control neurons. An AAV is known as a “vector” for gene therapy. This means scientists can insert desired DNA into the AAV allowing it to deliver and express these genes to certain cells in the body. [8] In ATAC, FUS-BBBO first opens specific areas in the BBB for AAV to deliver its genes. In this way, FUS-BBBO essentially “marks” the specific neural circuits to be treated, contributing to the spatial specificity of the ATAC treatment. The AAV vectors contain DNA that codes for engineered proteins called DREADDs, a type of cell receptor. These cell receptors allow a cell to respond to designer drugs, which are drugs created in the lab, that can get into -  but do not activate naturally-occurring receptors in - the brain. When the AAV vector delivers the DREADD genes to the specific neurons marked by the FUS-BBBO procedure, the expression of the DREADDs allows the neurons to be activated by designer drugs. The designer drug used in the ATAC procedure is called clozapine-N-oxide (CNO). CNO has the ability to excite or inhibit certain neurons that express DREADDs, allowing for targeted treatment of neurological disorders. Furthermore, it can be administered in a non-invasive way to patients (i.e., as a pill). [9] It is the combination of FUS-BBBO, gene therapy through the AAV vectors, and CNO that makes ATAC so effective at targeting specific neurons. (Fig. 2)

Figure 2. Depiction of the ATAC procedure. [1]

The Szablowski lab chose to test ATAC by observing its effect on the hippocampus of rodents. Dr. Szablowski describes the hippocampus as “a very relevant brain region for many disorders - epilepsy being one of them. ” So, if ATAC is effective on the hippocampus, then it has the potential to treat a multitude of disorders, ranging from epilepsy to PTSD. Specifically, initial tests focused on how ATAC could modulate memory formation, providing a possible treatment for anxiety disorders. To test the efficacy of the treatment, rodents were exposed to a fear-inducing stimulus in a unique experimental setting. Next, both the rodents who had received AAV and DREADDs and those in a control group received either CNO or saline solution. Twenty-four hours after initial exposure to the stimulus, the rodents were again placed in the same experimental setting to test their fear recall.

The results suggest that ATAC applied in the hippocampus successfully reduced memory formation. In a common memory task, ATAC rodents that received CNO showed less than half of the response than rodents in the control group. Furthermore, the results showed ATAC only targeted memory formation and had no effect on exploratory behavior and sensation of stimuli, confirming the high specificity of the treatment. What does this all mean for us? Well, if ATAC becomes clinically relevant, it could enable a minimally invasive, highly effective way of treating neurological or psychiatric disorders including Parkinson’s disease or epilepsy. As Dr. Szablowski describes, “any [neuropsychiatric disorder] that has the defined activity of the [neural] circuit that needs to be modified” could potentially be treated with ATAC. 

In practice, Dr. Szablowski envisions that ATAC would first involve an outpatient procedure that opens the BBB and delivers the gene therapy. After waiting about three weeks, the patient would simply take pills regularly to deliver the CNO to the “painted” areas of the brain. There would no longer be a need for implanted devices and multiple intracranial injections. It’s a streamlined, powerful form of treatment that can greatly simplify what a patient must go through while seeking relief from neurological disorders. However, ATAC still faces a long journey toward reaching clinical relevance. Like all biomedical research endeavors, ATAC must overcome many obstacles. For example, Dr. Szablowski recognizes that ATAC would be a very expensive procedure with current technology and techniques. So, his lab is currently developing accessory technology for ATAC, such as “site-specific therapeutics,” a technique that can be used before AAV vectors to temporarily modulate a specific site of the brain. This would allow medical practitioners to test whether or not their chosen target in the brain for ATAC is indeed correct, based on the disorder and the individual. Site-specific therapeutics would help prevent the unfavorable situation in which expensive, irreversible, and potentially risky gene therapy has been applied to the incorrect site in the brain during ATAC. Once ATAC is ready for  clinical trials, they’ll start testing this combination of site-specific therapeutics and ATAC for the treatment of Parkinson’s disease.

With careful planning and creative thinking, the Szablowski lab has created a minimally invasive neurological disorder treatment that maximizes effectiveness and simplifies procedures. While ATAC faces and will continue to face obstacles on its path toward implementation in the medical field, this unique solution brings us closer to a future of accessible and optimized biological treatments for complicated disorders.

Works Cited

[1] Szablowski, J. O.; Lee-Gosselin, A.; Lue, B.; Malounda, D.; Shapiro, M. G. Acoustically Targeted Chemogenetics for the Non-Invasive Control of Neural Circuits. Nature Biomedical Engineering 2018, 2 (7), 475–484. 

[2] Kozai, T. D. Y. The History and Horizons of Microscale Neural Interfaces. Micromachines 2018, 9 (9), 445. 

[3] Kim, C. Y.; Ku, M. J.; Qazi, R.; Nam, H. J.; Park, J. W.; Nam, K. S.; Oh, S.; Kang, I.; Jang, J.-H.; Kim, W. Y.; Kim, J.-H.; Jeong, J.-W. Soft Subdermal Implant Capable of Wireless Battery Charging and Programmable Controls for Applications in Optogenetics. Nature Communications 2021, 12 (1). 

[4] 2021 Autumn Inside NIMH. https://www.nimh.nih.gov/research/research-funded-by-nimh/inside-nimh/2021-autumn-inside-nimh (accessed Apr 6, 2022). 

[5] Gooch, C. L.; Pracht, E.; Borenstein, A. R. The Burden of Neurological Disease in the United States: A Summary Report and Call to Action. Annals of Neurology 2017, 81 (4), 479–484. 

[6] Daneman, R.; Prat, A. The Blood–Brain Barrier. Cold Spring Harbor Perspectives in Biology 2015, 7 (1). 

[7] Wang, J. B.; Di Ianni, T.; Vyas, D. B.; Huang, Z.; Park, S.; Hosseini-Nassab, N.; Aryal, M.; Airan, R. D. Focused Ultrasound for Noninvasive, Focal Pharmacologic Neurointervention. Frontiers in Neuroscience 2020, 14.

[8] Naso, M. F.; Tomkowicz, B.; Perry, W. L.; Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 2017, 31 (4), 317–334.  

[9] Burnett, C. J.; Krashes, M. J. Resolving Behavioral Output via Chemogenetic Designer Receptors Exclusively Activated by Designer Drugs. The Journal of Neuroscience2016, 36 (36), 9268–9282.

Evaluating the Implications of COVID-19 on Patients with Sickle Cell Disease

by Harveen Kaur

Abstract

The presence and rapid spread of the novel infectious disease COVID-19, caused by the respiratory pathogen/coronavirus SARS-CoV-2, has encompassed the world as a global pandemic. COVID-19 spreads contagiously through air and droplets of bodily fluids, such as saliva, and has an array of symptoms associated with its contraction and spread, leading to approximately 364 cumulative coronavirus-related hospitalizations per 100,000 individuals only in the United States during early 2021. Additionally, COVID-19 cases are often more severe in individuals that have pre-existing medical conditions, with sickle cell disease being at the top of the list. Sickle cell disease, also referred to as sickle cell anemia, is an inherited blood disorder that abnormally distorts the structure and function of hemoglobin in the body leading to oxygen deficiency. This medical condition has been associated with increasing the risk of developing a severe case of COVID-19, but a definite connection has not yet been confirmed that categorizes the risk factor for various age groups. Studies published in September 2020 report that an international registry, called SECURE-SCD, has been created to track patients suffering from both conditions and their respective health outcomes. By using the data from various documented studies, this article hypothesizes that there is an important connection between sickle cell anemia and COVID-19 that leads to enhanced symptoms and overall higher fatality in patients suffering from the two. Hence, the primary purpose is to dissect and evaluate the specific linkage between the SARS-CoV-2 coronavirus and sickle cell anemia including the risk factors for both medical conditions experienced simultaneously and based on key demographic categories. Establishing the connection between sickle cell disease and COVID-19 is significant to better understand the implications of the virus for those with pre-existing conditions and potentially adjust treatment protocols that are currently being discovered.

Introduction

Infectious diseases have proven to be the ‘silent killer’ of the world throughout history. Ranging from the Black Death bubonic plague, the Spanish flu, and the swine-origin influenza A (H1N1) virus, the slow but steady upward  trend of infectious disease occurrence  enforces the fact that a human-dominated world can never thrive with imbalances in nature [1]. Many of these infectious diseases are zoonoses, diseases in animals that are transmitted to human beings, while others are classified as viruses transmitted by human beings to other human beings [2]. Virulent microorganisms have been present  among humankind for thousands of years, but it is only within the past couple of centuries that they have caused a huge leap in mortality. The most recent example of a globally destructive pathogen is the SARS-CoV-2 coronavirus, which causes the respiratory syndrome [2]. 

COVID-19, a respiratory illness that the world has been facing as a pandemic for over one year, is a contagious infectious disease stemming from the SARS-CoV-2 respiratory pathogen with the most common symptoms as fatigue, cough, and fever [3]. According to the CDC, the virus primarily spreads through respiratory droplets that arise through coughing and/or sneezing [3]. However, recent studies and speculation have stated that transmission may be airborne due to the presence of SARS-CoV-2 viral RNA in air samples within droplet nuclei that stay infectious if left in the air over long distances and large amounts of time [4]. Currently, there is no definitive cure for COVID-19, but many countries including the United States have implemented methodologies to track and contain the virus, especially through vaccine development. Specifically, roughly 78 vaccines for COVID-19 are undergoing clinical trials, with seven vaccines (such as those developed by Pfizer-BioNTech and Moderna) already distributed for full use [5]. These highly infected countries have also encouraged individuals to socially distance themselves from others by at least six feet and wear masks, both of which help control daily cases and fatalities from COVID-19 [6]. Despite these preventative measures, the implications of COVID-19 have affected millions of individuals in the United States and on a global scale. Vulnerable populations that already suffer from pre-existing medical conditions are at high risk to develop and contract a severe case of COVID-19. One such diagnosis that has caused an uproar in the United States is sickle cell disease.

Sickle cell disease (SCD), also called sickle cell anemia, consists of a group of inherited disorders that changes the shape of red blood cells from disc-shaped to crescent-shaped when atypical hemoglobin is present [7]. Through this change, red blood cells are more prone to blocking blood flow and do not move easily in the body, which can lead to an onset of chronic conditions such as strokes and other pain crises [7]. With the spread of COVID-19, however, those suffering from SCD have been studied to have a higher rate of contraction, as well as more severe symptoms and outcomes when coupled with the virus [8]. For example, SCD patients with COVID-19 have a higher overall case fatality (10.9% fatality rate) than non-SCD individuals contracting the virus (3.3% fatality rate) [9]. Patients suffering from SCD have cardiopulmonary comorbidities that COVID-19 heightens, but the specific linkage between the two has yet to be closely compared. However, certain unmistakable trends connecting patient’s age, race and severity of SCD symptoms to their COVID-19 prognosis have been observed [8]. This article focuses primarily on the results from various national and international registries that collect information about COVID-19 and SCD by comparing the effects of suffering from both conditions together, based on age groups and other patient demographics. Establishing and characterizing the connection between the two conditions can create a more accurate solution on how to protect oneself from COVID-19 when suffering from SCD.  

Materials/Methods

The connection between SCD and COVID-19 transmission and symptoms, when specifically focusing on age and race, can be outlined through the data found in three studies. 

The first study, published on the Centers for Disease Control and Prevention, was conducted from March to May 2020 in the United States and was recorded in the Medical College of Wisconsin SECURE-SCD Registry [10]. This particular registry examines COVID-19 cases among those living with SCD in the United States, and the results of the study were limited to a two-month time period in which such patients were analyzed and documented. The study primarily focused on the average age of these patients, the number of SCD-related complications these patients have had in the past, and the resulting intensity of their current COVID-19 symptoms [10].

Another study, published in the journal Haematologica, referenced 10 patients in the UK who had contracted SARS-CoV-2, and all 10 patients had hemoglobin SS disease, which is the most common and most severe type of SCD [11]. The severity of this particular kind of SCD also means that these patients experience the worst symptoms at the highest rates for the condition [12]. With the most recent documentation being published in November 2020, the recovery rates of these patients were analyzed. The figure below from the same study highlighted the steps of evaluation conducted for the patients with SCD who had symptoms that aligned with potentially contracting COVID-19 as well as further standards of treatment and evaluation based on individual patient status [11]. The diagram shows the process of triage for patients with SCD who also contracted COVID-19 based on symptoms and patient accessibility [11].

Figure 1 Determining the appropriate medical response for SCD patients with COVID-19, ranging from patient instructions, types of medical evaluation, and potential therapies [11].

A third study, presented at the American Society of Hematology, was led by Dr. Lana Mucalo from the Medical College of Wisconsin and required the examination of 370 COVID-19 cases in patients already suffering from SCD from an international sickle cell registry [13]. This particular study compared the data found from the patients listed in the international registry to patients suffering from both conditions in the general Black population, keeping race as the measured demographic independent variable [13]. 

Results

The results of the first study, documented by the Medical College of Wisconsin SECURE-SCD Registry in between March 20, 2020 and May 21, 2020, showed that roughly 178 individuals who had SCD — and who were not simply carriers of the sickle cell trait — also contracted COVID-19 during this time. [10] Out of these 178 individuals, 7% (13 individuals) died upon contracting COVID-19 and being hospitalized, and 11% (19 individuals) were admitted to the intensive care unit while suffering from both conditions simultaneously [10]. More than half of the total individuals had suffered an SCD-related complication in the past that led to prior hospitalizations, and the average age of the 178 individuals at the time was roughly 28.6 years [10]. On the contrary, individuals with COVID-19 that don’t suffer from SCD have a death rate of less than 1% for those 20—54 years of age in early 2020, which is dramatically less than rates analyzed for patients with both SCD and COVID-19 [14].  

From the 10 patients in the UK documented in Haematologica, a 54-year-old patient died who suffered from delayed hemolytic transfusion reactions and also had high levels of CRP (C-reactive protein), a marker of negative prognostics for patients suffering from COVID-19 [11]. From all 10 patients, two underwent hydroxyurea therapy, seven had regular blood transfusions, and a total of five patients were managed remotely through telephone contact for their treatment [11]. Medical professionals and experts believed that suffering from the SARS-CoV-2 infection along with SCD would lead to acute chest syndrome (ACS). However, in the 10 patients, only one of them suffered from related respiratory complications; this was the same patient who passed away during clinical monitoring [11]. 

In the final study, the Black population was examined since, especially in the United States, individuals with African-American descent are statistically more likely to have both the sickle cell trait as well as the disease [13]. Additionally, completely separate from SCD, Black Americans are nearly four times more likely to be hospitalized due to COVID-19 as the virus has progressed globally [13]. Using race as the measured variable, this study concluded that those with SCD are roughly 6.2 times more likely to suffer a fatal complication from COVID-19 when compared to the general African-American population in the United States [13]. While the fatality rate based on ages 18-34 and 35-50 for the general Black population is less than 1% for all Black people in both age groups, those with SCD had death rates of 2.6% and approximately 12%, respectively, for the same age groups [13]. Additionally, a significantly larger amount of COVID-19 hospitalizations for Black individuals with SCD occurred among the younger age group, which correlates with larger death rates as age decreases [15].

Discussion

With the knowledge of all three pieces of data, these results illustrate that there is a correlation regarding age and race when examining the compounding effects of SCD and COVID-19. When examining the whole percentage of individuals who were hospitalized and severely affected by suffering from both conditions, the data from the SECURE-SED Registry suggests that such a connection exists. Additionally, data from the registry includes reports of 7% fatalities and 11% admittance to the ICU over 60 days for individuals with both conditions, which is heavily alarming. Even in the UK, the data suggests that there is an evident conjunction between the two conditions, despite the study being conducted with a small sample size. If 1 out of 10 individuals died, a 10% mortality rate from the combined detrimental effects of both conditions is just as concerning, if not more, than the first study. Lastly, the final study examined the effects of racial background on contracting COVID-19 while having SCD, and a significant relationship between the two is evident. In fact, having SCD, particularly for Black Americans, dramatically increases the fatality rate of these individuals from COVID-19 (by up to 11%). Based on these aforementioned studies, strong evidence exists to suggest that SCD and COVID-19 do have detrimental implications for individuals on a global level.

Conclusion

After understanding the symptoms of patients already suffering from SCD, adding the effects of COVID-19 can exponentially deteriorate a patient’s health. Through the three different pieces of data, there is a noticeable connection between incidence of SCD and severity of symptoms of COVID-19, the most recent and prevalent globally-spread virus. These connections can be analyzed on the basis of age as well as race, thereby indicating that the hypothesis for a linkage between the two conditions is well-supported. Since COVID-19 treatments and vaccinations are still being actively manufactured, knowing its added implications for those with SCD can be significant when determining a proper healing regimen for the virus. Additionally, understanding the damage done by both ailments combined can help reduce their high mortality rate, thereby creating more successful outcomes for patients for both diseases individually and together. 

Works Cited

[1] Bean, Mackenzie. Becker’s Hospital Review. 2020. 

[2] Tabish, Syed Amin. International Journal of Health Sciences. 2009, 3(2) V-VIII.

[3] Coronavirus Disease 2019 [Online]. 2021. https://www.cdc.gov/dotw/covid-19/index.html (accessed Mar. 17, 2021).

[4] Liu, Jiaye. Emerg Infectious Dis. 2020, 26(6), 1320-1323.

[5] Zimmer, Carl. The New York Times. 2021.

[6] Thu, Tran P. B. Elsevier Pub Health Emerg Cond. 2020, 742.

[7] National Heart, Lung and Blood Institute. US Dept. of Health & Human Services. 

[8] Shet, Arun. American Society of Hematology. 2021.

[9] Minniti, Caterina P. Blood Advances. 2021, 5(1), 207-215.

[10] Panepinto, Julie A. Centers for Disease Control and Prevention. 2020.

[11] Menapace, Laurel A. Haematologica. 2020, 105 (11), 2501-2504.

[12] Rogers, Graham. Healthline. 2019. 

[13] Thompson, Dennis. Medical Xpress. 2020

[14] Razzaghi, Hilda. Centers for Disease Control and Prevention. 2020, 69(12), 343-346.

[15] Mucalo, Lana. American Society of Hematology. 2020.

Evaluating the effectiveness of CQ and/or HCQ against SARS-CoV-2

by Puneetha Goli

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). Patients exhibit a spectrum of severity in their symptoms ranging from asymptomatic to breathing difficulties[1]. The purpose of this article is to evaluate the effectiveness of chloroquine (CQ) and its less toxic metabolite hydroxychloroquine (HCQ) as a viable treatment for SARS -CoV-2 through analyzing data from available research studies. In one of the early breakthrough studies published in February 2020, Gao et al. found that a low-micromolar concentration of CQ blocked COVID-19 infection in early in vitro studies and improved lung imaging, indicating a potential correlation between CQ administration and a decrease in the spread of COVID-19[2]. However, more recent studies, including Skipper et al., since then have indicated that there are no significant differences with the administration of the agents, and some even caution against the use of them because of potential long-term side effects [3,4]. The general consensus among the scientific community, however, remains that the use of agents must be rigorously considered on a case-by-case basis and with caution in research or clinical trial settings[4]. Determining the effectiveness of CQ/HCQ will aid in preventing the spread of misinformation. 

Introduction 

Responsible for taking the lives of 2,244,713 people worldwide (as of February 3, 2021), the World Health Organization (WHO) classified COVID-19 as a pandemic in March 2020 (5,6). SARS-CoV-2, the virus responsible for COVID-19, is only one of seven identified human coronaviruses and is primarily transmitted from person-to-person through direct contact, saliva, and airborne droplets. It is considered “novel” because it is newly identified and is different from the other coronaviruses that have been linked with causing mild illnesses, such as the common cold [7]. Because of its novel nature, there is much that is unknown about the virus and the disease, and unfortunately misinformation regarding treatments has been spread, specifically the effectiveness of chloroquine (CQ)/ hydroxychloroquine (HCQ) as a treatment [7]. 

CQ and HCQ are antimalarial treatments, and interest in these drugs as possible treatments for COVID-19 was spurred in part from the drugs’ history of in vitro activity against viruses such as influenza. HCQ’s role in modulating the immune system through treating other autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis, could have also played a role [4,7].  

In April 2020, nearly 55 countries, including the US, Mauritius, and Seychelles, were in the process of receiving HCQ from India [8]. Country leaders, including Brazil’s President Jair Bolsonaro and United States’s President Donald Trump, showed support for the drug, some even claiming to have taken it [9,10]. 

In the months since, however, scientists and national agencies have discouraged the use of CQ and HCQ against treatment for COVID-19, claiming that its effectiveness hasn’t been proven yet. Evaluating the research surrounding CQ and HCQ’s effectiveness is critical in order to address misconceptions and misinformation that is being spread as well as to evaluate its role as a potential treatment.

This paper will be reviewing two studies presenting contradictory evidence regarding the effectiveness of CQ/HCQ. In the first study published on March 16, 2020, Gao et al. examines the effectiveness of CQ in the early stages of the COVID-19 pandemic. Later, a study published on October 20, 20202 by Skipper et al., provides a more comprehensive comparison documenting HCQ’s utility.

Materials and Methods 

Gao et al., one of the early breakthrough studies which found favorable results in treating pneumonia associated with COVID-19 with CQ/HCQ, first conducted in vitro studies in which CQ was tested against the COVID-19 infection [2]. After a determination of a half-maximal effective concentration (EC50) and a half-cytotoxic concentration (CC50), multiple clinical trials were then conducted in China.100 patients from more than 10 hospitals in Wuhan, Jingzhou, Guangzhou, Beijing, Shanghai, Chongqing, and Ningbo were treated with chloroquine phosphate and results, including lung imaging findings, were gathered. 

The following section examines the method used in Skipper et al, which found that HCQ did not significantly decrease the severity of symptoms among patients experiencing early, mild COVID-19[3]. The study is the first randomized clinical trial studying COVID-19 treatments among outpatients with early, mild COVID-19 through a randomized, placebo-controlled procedure.

Participants

Participants included non-hospitalized, symptomatic adults that either were lab-tested for COVID-19 or those who demonstrated COVID-19 compatible symptoms and had an epidemiologic link to a lab-confirmed COVID-19 contact. Due to the scarcity of COVID-19 tests during the time the study was conducted, not all of the participants were tested for COVID-19, and the second criterion had to be included to recruit subjects. Further, adults were only enrolled if they had experienced COVID-19 symptoms for 4 or fewer days.

A total of 491 subjects were enrolled in the study (244 assigned to HCQ and 247 assigned to placebo), however only 423 contributed toward data collection (231 assigned to HCQ and 234 assigned to placebo) because they had completed at least 1-follow up survey with symptom data. 

 14-Day Trial

Participants were given either 200-mg tablets of HCQ sulfate or masked placebos of either 400mcg folic acid or lactose by a research pharmacist. Both the HCQ and placebo shared similar physical characteristics and opaque dispensary bottles so that the two tablets were hard to distinguish between. 

On the first day, adults in the experimental group first took 800mg of oral HCQ (4 tablets) and an additional 600mg (3 tablets) after 6 to 8 hours. For 4 more days after, participants continued to take 600 mg daily (for a total of 5 days). Participants in the placebo group were prescribed to take the placebo tablets in a similar regiment. The dose was based on previously published pharmacokinetic measures that serve to maintain the HCQ concentration above the EC50 for SARS-Co-V-2. 

Outcomes and Data Collection

Researchers collected data regarding symptoms and severity through participant-reported surveys on day 1 (baseline), 3, 5 (end of medication), 10, and 14. A 10-point visual analogue scale was used to collect data regarding symptom severity. The study’s primary endpoint was a measure of overall change in symptoms throughout the study’s 14-day period.

Results 

Although both Gao et al. and Skipper et al. examine the effectiveness of CQ and/or HCQ against SARS -CoV-2, the studies report different experimental approaches and results

While Gao et al. didn’t publish the exact data and figures for their study, they concluded that the patients treated with CQ showed better results in inhibiting the exacerbation of pneumonia, facilitating an environment for virus-negative conversion, improving lung imaging, and reducing the disease course [2]. Researchers also noted that no severe adverse reactions as a result of the CQ were found among the 100 patients.

In the Skipper et al. study however, differences in data collected between the placebo and HCQ were not significant [3]. As depicted in Figure 1, data on day 5 of the study showed that 54% of participants in the HCQ group reported symptoms versus 56% in the placebo group. Further, at day 14, 24% in the HCQ group reported symptoms compared to 30% in the placebo group. The study concluded that the proportion of participants that reported symptoms was not significantly different between the HCQ and placebo group.

Figure 1. Percentage of participants reporting COVID-19 symptoms throughout the Skipper et al. study’s 14-day time period [3]. On day 5, the percentage of HCQ participants with symptoms was 2 points less than that of the placebo participants, and by day 14, the difference had increased to 6 points, but still not enough for statistical significance.

In addition to the proportion of COVID-19 symptoms observed in both groups, Skipper et al. recorded the severity of the symptoms based on a 10-point visual scale. As shown in Figure 2, the participants in the HCQ group reported an average reduction of symptom severity of 2.60 throughout the 14 day period, while placebo participants reported a 2.33-point reduction. Although the HCQ group saw an overall 12% more reduction in symptom severity compared to the placebo group, the difference was, once again, not statistically significant.

Figure 2. Overall scores of symptom severity throughout the Skipper et al. study’s 14-day time period [3]. Although the data showed a 12% relative reduced severity difference for the HCQ in comparison to the placebo, it was not statistically significant.

Discussion

Gao et al. is one of the earliest studies that showed favorable results for CQ. The researchers have hypothesized that evidence for the CQ’s anti-viral properties lies in previous studies which have found the drug to increase the endosomal pH needed for the fusion of the virus. In addition the drug is suspected to cause interference with the glycosylation of the SARS-CoV cellular receptors [11,12]. Based on these findings along with the timing of the study (before the WHO classified COVID-19 as a pandemic), the researchers recommended CQ be used to treat patients that experience pneumonia associated with COVID-19 in China.

Therefore, although Skipper et al. points out differences among the HCQ and placebo groups, they weren’t statistically significant, contradictory to the findings of Gao et al.

Conclusion

Differences in results across studies, as sampled through Gao et al. and Skipper et al., have led scientists to continue to question and rethink the effectiveness of HCQ/CQ against COVID-19. While a concrete conclusion regarding the drugs’ fates is yet to be determined, scientists continue to caution the use of HCQ/CQ for public use and rather recommend the drug to preferably only be administered under carefully designed clinical trials, as well as examined on a case-by-case basis [4]. 

With vaccinations becoming more widely available, discussion and debate centered around the administration of HCQ/CQ has largely reduced, and rather, the focus has shifted towards the effectiveness of the vaccines. Although a 2021 study has found the Moderna vaccine to be antibody persistent through 6 months, there isn’t exact data regarding the maximum efficiency of this particular vaccine as well as that for the Pfizer-BioNTech and Johnson & Johnson vaccines, the two others approved by the Food and Drug Administration [13]. Future directions of study can involve a close examination of these vaccines. Results and information collected from the close examination of these vaccines would be critical in planning the continued management of the COVID-19 pandemic in the United States. 

References 

[1]Transmission of SARS-CoV-2: implications for infection prevention precautions. World Health Organization [Online], July 9, 2020, https://www.who.int/news-room/commentaries/detail/transmission-of-sars-cov-2-implications-for-infection-prevention-precautions (Feb. 3, 2021)

[2] Gao, J. et al. BioSci. Trends 2020, 14, 72-73

[3] Skipper, C. P. et al. Ann. Intern. Med 2020, 173, 623-631

[4] Meyerowitz, E. A. et al. FASEB 2020, 34, 6027-6037

[5]World Health Organization. https://www.who.int/ (accessed Feb. 3, 2021)

[6]Archived: WHO Timeline - COVID 19. World Health Organization [Online], Apr. 27, 2020, https://www.who.int/news/item/27-04-2020-who-timeline---covid-19 (accessed Feb. 3, 2021)

[7] Centers for Disease Control and Prevention. https://www.cdc.gov/ (accessed Feb. 3, 2021)

[8] India sending hydroxychloroquine to 55 coronavirus-hit countries. The Economic Times, April 16, 2020. https://economictimes.indiatimes.com/news/politics-and-nation/india-sending-hydroxychloroquine-to-55-coronavirus-hit-countries/articleshow/75186938.cms?utm_source=contentofinterest&utm_medium=text&utm_campaign=cppst (accessed Feb. 3, 2021)

[9]Stargardter, G.; Paraguassu, L. Special Report: Bolsonaro bets 'miraculous cure' for COVID-19 can save Brazil - and his life. Reuters, July 8, 2020. https://www.reuters.com/article/us-health-coronavirus-brazil-hydroxychlo/special-report-bolsonaro-bets-miraculous-cure-for-covid-19-can-save-brazil-and-his-life-idUSKBN249396 (accessed Feb. 3, 2021)

[10] Bruggeman, L. Hydroxychloroquine returns as wedge between President Trump, health advisers. ABC News, July 28, 2020. https://abcnews.go.com/Politics/hydroxychloroquine-returns-wedge-president-trump-health-advisers/story?id=72036996 (accessed Feb. 3, 2021)

[11] Savarino, A. et al. Lancet Infect Dis. 2003, 3, 722-727

[12] Yan, Y. et al. Cell Res. 2013, 23, 300-302

[13] Doria-Rose, N. et al. NEJM. 2021

Determining the Efficacy of Sourcing IgY Antibodies from Chicken Eggs Using the PierceTM Chicken IgY Purification Kit

by Sachi Kishinchandani

ABSTRACT

IgY is known to be an immunoglobulin that is formed by the maternal immune system and passed down to offspring, in reaction to certain foreign substances. It is hypothesized that IgY can be used to boost human immune systems. Thus, to ask how we can easily gather these proteins for increased human immunity, we analyzed the purification of IgY via the Thermo ScientificTM PierceTM Chicken IgY Purification Kit. Through a Bradford assay, SDS PAGE, and a Densitometry, we determined that it is possible to purify around 40-80 mg of IgY per egg using similar methods. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible.

INTRODUCTION/BACKGROUND

Our immune system plays a vital role in protecting our body from foreign diseases, viruses, and harmful bacteria. Therefore,the study of its mechanisms and functions is extremely important. One important feature of the immune system across a variety of species is how antibodies are passed from mothers to their newborn offspring [1]. Over time, natural selection has led to adaptation of antibodies to be passed down to offspring for survival, whether it is in the form of the IgG antibodies passing through the placenta in mammals, or birds passing down IgY antibodies in their egg yolk [1].

By studying the IgY antibodies found in chicken eggs, we can study and potentially introduce useful IgY antibodies into humans (for example, via the consuption of IgY Antibodies), in order to bolster our immune systems. IgY antibodies can be used to create immunization in a form that does not specifically require a vaccine [2]. People can be passively immunized against respiratory illness with the help of IgYs to help neutralize respiratory pathogens and disease spread. IgY is an ideal candidate for this antibody insertion due to its structural similarity to the IgG antibodies common in humans, as well as its ability to target specific antigens [3]. IgY has been found to bind specifically to influenza virus Nucleoprotein (NP) by detecting multiple virus subtypes in swine [4]. These traits make IgY a worthwhile subject of study, especially one whose potential outcomes are so beneficial. 

In this series of experiments, we focused on investigating IgY protein concentrations, abundance, and purity in potential sources for IgY protein purification, as the extent of these properties are not widely available in current literature. We used the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to extract the IgY protein from the egg yolk [5]. The extraction of the IgY protein allowed us to conduct a Bradford assay, SDS PAGE, and a Densitometry in order to determine the concentration, abundance and purity of the protein. With the results of this experiment, we determined that we can effectively purify IgY from store-bought chicken eggs.

MATERIALS AND METHODS

PHYSICAL PURIFICATION OF IgY PROTEIN

We purified IgY from store-purchased Eggland Best eggs using the Pierce™ Chicken IgY Purification Kit from Thermo Fisher to physically separate the antibody rich yolk from the white using a series of suspensions and centrifugations with reagents. We conducted this by first separating the egg yolk from the egg whites. We then rolled the egg yolk on a paper towel to remove excess egg-white protein, pricked the yolk sac and then drained an approximate 8 mL of the yolk into a beaker. This step is essential to remove any contaminant protein from the egg white. We then added five times the egg yolk volume of cold Delipidation Reagent (40 mL) from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to the 8 mL egg yolk. The mixture was stirred until well incorporated and then stored at 4 ºC for a week. 

The mixture was then centrifuged for 15 min at 6,000 × g in a refrigerated centrifuge. The colorless and translucent supernatant was decanted and placed in a secondary conical tube, to which the precipitation reagent solutions from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit were added. The mixture was placed in a 4 ºC incubator overnight, and then centrifuged for 15 min at 6000 rpm in a 4 ºC refrigerated centrifuge. We then isolated the pellet from the conical tube.

Our final step in the purification of the protein was to resuspend the pellet in Phosphate buffered saline (PBS) (100 mM Na3PO4, 150 mM NaCl, pH 7.2). We added the PBS to the pellet and stirred the mixture until it was well incorporated. 

BRADFORD ASSAY

Our next step was determining the concentration of the IgY protein using a Bradford assay. The assay uses acidic Coomassie dye to bind to proteins and result in a visible color change from brown to blue, the intensity depending on the amount of protein present. We measured out 5 mL of Bradford reagent into 8 glass test tubes. We pipetted Bovine Serum Albumin (BSA) protein into the first six test tubes, and IgY into the remaining two test tubes. With the BSA protein test tubes, we measured the absorbance at 595 nm for each solution with a spectrophotometer, and created a standard curve of the absorbance in relation to BSA protein amounts. We then measured the absorbance of our protein to be 0.383. 

SDS PAGE

Because current literature information on IgY proteins states that two chains of IgY should occur around 70 kDa and 30 kDa [6], we decided to make our gel a 12% acrylamide. With our SDS PAGE, we first created a standard mixture of 1 L Running buffer, 10 mL of 1x Separating buffer and 10 mL 1x Stacking buffer. We also created a 1 mL of 10% APS solution for activating the polymerization of the gel by dilution of stock APS available in the lab. We then added 100 uL of APS and 10 uL of TEMED into both the Stacking and Separating buffers. We then began to pipette the Separating buffer solution in between the glass plates until a marked line, allowing the Separating buffer to set with isopropyl alcohol before adding the Stacking buffer on top. While the Stacking buffer was still liquid, we inserted the comb to create the wells in the gel. 

We stored the gel for four days in a moist paper towel, then conducted SDS PAGE with 5 uL of our protein and 5 uL of sample buffer in each of our wells, with the Broad Range 5 Microliters NEB in one of the wells for reference. We let the SDS PAGE run for 90 min --the first 10 min at 120 V and the rest of the time at 180 V--until the bands reached the bottom of the gel, and then stained the gel with Coomassie blue solution. We then destained the gel using the premade solutions Destain #1 (50% methanol, 10% acetic acid) and Destain #2 (7% methanol, 10% acetic acid). 

DENSITOMETRY

This part of the experiment was to determine the concentration of the IgY bands. The densitometry was conducted in a similar fashion to the SDS PAGE. In order to address the possible presence of a dimer in our previous SDS-PAGE gel, we increased the concentration of BME in two of the wells with our IgY protein in the hopes of removing any remaining disulfide bonds. Instead of creating a gel for this experiment, we used a BIO-TEC premade gel. We created 5 solutions of different concentrations of BSA: 0.2 ug, 0.5 ug, 1 ug, 1.5 ug, and 2 ug. We then added 5 uL of these solutions, mixed with 5 uL 2x Laemmli buffer into separate wells. We also added our purified IgY protein into the gel, with two wells made with 5 uL of protein and 5 uL 2x Laemmli buffer each, and two wells with 5 uL protein and 5 uL 10% BME Laemmli buffer each. We ran the gel at 300 V for 15 min, until the dye front hit the bottom of the gel. We stained and destained the gel exactly as we did for the SDS PAGE. We then used a machine to analyze the concentrations and Molecular Weight (MW) of the bands of IgY in comparison to BSA.

RESULTS & DISCUSSION

The overall purpose of the experiments was to determine the practicality of extracting large quantities of protein from store-bought chicken eggs. From the experiments, we were able to determine the following:

PHYSICAL IgY PURIFICATION

The weight of our purified protein match the expected result of using the kit. We were able to purify 70.2756 mg of IgY solution from a single store-bought egg (Table 1).

Table 1. The total mass of IgY protein solution as a result of the initial purification of IgY protein with Thermo ScientificTM PierceTM Chicken IgY Purification Kit. 

BRADFORD ASSAY

Once our protein was purified, we were able to determine the concentration of the protein from a Bradford assay, comparing the absorbance of our protein to the absorbance of BSA. 

Figure 1. The absorbance of BSA over the concentration of BSA measured via a spectrophotometer, used as a standard curve to determine that the concentration of our IgY protein is 2.43 ug/uL.

Table 2. Calculated concentrations of our IgY solution as determined by a Bradford Assay. Conducted by measuring the absorbance of different volumes of the IgY solution in a spectrophotometer. These data were used to find the average concentration of the IgY protein to be 2.43 ug/uL. 

With these calculated concentrations (Table 2), we found that the average concentration of our IgY sample is 2.43 ug/uL. This matched expected results as previous studies have estimated that the concentration of IgY purified from egg yolk should be 2-4 mg/mL [7]. 

SDS-PAGE

Knowing the concentration of the protein helped us conduct SDS-PAGE, which would help us find out the size and purity of our purified IgY protein. 

Figure 2. SDS-PAGE of our IgY protein with clear bands around 27 kDa and 130 kDa. Done in a 12% acrylamide gel that was stained with Coomassie blue solution. The left-most well on the right end of the figure indicates standard protein molecular weights from the NEB Protein standard solution. The remaining wells have 5uL of 2.34 ug/uL of IgY and 5uL of 2X Laemmli buffer, as indicated at the bottom of each column in the figure.  

Figure 3. Graph of the log (molecular weight) of NEB Protein Standard as a function of relative distance. The curve between the distances 2.5 and 4.4 cm was used to find the MW of the heavy band to be 117.7 kDa. The curve between the distances 14.7 and 18.6 cm were used to find the MW of the second band to be 27.28 kDa.

Once we conducted our SDS-PAGE and stained/destained it, we found that the gel showed significant IgY purity because the most significant bands were at around 27 kDa and 130 kDa. We also found a light band around 43 kDa (Figure 2), which was confirmed to be the contaminant ovalbumin by our instructors (see acknowledgments). 

We calculated the MW of the bands to be 27.28 kDa and 117.7 kDa. In order to do so, we calculated the equation of the line between the points 2.5 and 4.4 cm to be logMW = -0.7169 x + 2.2932. When we plug 3.1 in for x, the MW ends up being 117.7 kDa. We also calculated the equation of the line between points 14.7 and 18.6 cm to be logMW = -0.02987x + 1.971. Plugging in 17.9 for x, we get MW = 27.28 kDa (Figure 3). 

We expected to have the band at 27.28 kDa for the IgY light chain, but our other expected value for the IgY proteins was around 70 kDa for the heavy chain, not 117.7 kDa, based on current literature on IgY. Because this 117.7 kDa band was almost exactly twice the weight of our expected heavy chain weight, we hypothesized that our protein’s disulfide bonds had not broken down completely. In order to understand whether this high molecular weight was a result of the small amount of BME in the 2x Laemmli solution, or was simply a contaminant, we conducted a densitometry with two of the wells containing our protein along with a higher concentration of BME. 

DENSITOMETRY

Figure 4. The results of our Coomassie stained and de-stained IgY and BSA Densitometry [left-most well: NEB marker. Well 2-3: 10% BME + IgY. Well 4-5: IgY + normal Coomassie. Wells 6-10: decreasing concentrations of BSA] The red arrow points to the heavy IgY chain, which is a band we did not observe in the previous (SDS PAGE) experiment. The blue arrow points to the light IgY chain. The yellow arrow points to the BSA chain. 

SDS-PAGE was run, this time with more BME and varied BSA amounts to both analyze the light/heavy chains and to possibly get a reading of the bands that would help us determine the concentration of protein in each band. Figure 4 shows there is clearly a large amount of BSA in wells 6-10, which would have been difficult to analyze our IgY density with, so we were unable to carry out analysis of the IgY. 

We found that the gel with the higher percentage of BME had a more prominent band around 72 kDa, along with bands around 27 kDa and 130 kDa. The finding of a band around 72 kDa and 27 kDa was consistent with current literature on IgY protein chains. The bands around 27 kDa and 130 kDa were consistent with our previous SDS PAGE (Figure 4). We still assume that the band around 130 kDa could be the IgY protein’s heavy chain that remains a dimer, however, there is a possibility that this could be a contaminant.  

This experiment does not tell us the identity of each of the protein bands found in the densitometry. If we had more time, we would have run the experiment again, using lower BSA concentrations and using the BSA band densities to make a standard curve in order to determine the total integrated density of the protein. From there, we would have calculated the density and concentration of the IgY, which would have told us the purity of the protein in relation to BSA. However, since we were unable to conduct this experiment, we suggest that further experiments should be performed to determine whether the IgY was effectively purified. 

CONCLUSION

Based on preliminary results, we can reasonably conclude that our IgY purification was successful and we were able to extract around 24.3 mg of pure IgY protein from the 8 mL of store-bought chicken eggs. Current literature indicates that the expected concentration of protein from each egg yolk is 2-4 mg/mL, which was what we observed in our results. Other studies using similar methods to our experiment have also found that it is possible to purify around 40-80 mg of IgY per egg using similar methods [7]. The reason we predict 40-80 mg per egg yolk is because we only extracted 8 mL of yolk, whereas the average egg yolk is about 15 mL, [8] so the total protein amount would be around 40 mg for 15 mL of yolk. The 24.3 mg of IgY extracted from one egg shows that eggs are a reliable source of IgY protein. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible. 

The extraction of IgY has important implications for further application. In the future, we hope to see multiple pathways for immunization built from IgY. One is the immunization of humans through digestion of IgY antibodies [9]. Another is epitope mapping, [10] identification and characterization of antibody binding sites on cells, which could help us understand the structures of antigen binding sites to combat diseases. The results of our purification of IgY seem promising for the future of IgY extraction and execution of further research. 

ACKNOWLEDGEMENTS 

We thank Dr. Thomas and Dr. Catanese for all their support and mentorship throughout the course. OURI, CUR, and the Biosciences Department for funding BIOS 211.  Anika Sonig and Aaron Lin for their partnership through this project and aid with the creation of the figures. BIOS 211 TAs for their guidance. 

Works Cited

(1) Pereira, E P V et al. International immunopharmacology 2019 vol. 73, 293-303. doi:10.1016/j.intimp.2019.05.015  

(2) Aymn Talat Abbas, Sherif Aly El-Kafrawy, Sayed Sartaj Sohrab & Esam Ibraheem Ahmed Azhar, Human Vaccines & Immunotherapeutics. 2019, 15:1, 264-275, doi: 10.1080/21645515.2018.1514224  

(3) Nagaraj et al. International Journal of Food Microbiology. 2016, 237, 136-141.

(4) da Silva M.C, Schaefer R, Gava D, Journal of Immunological Methods. 2018, 461, 100-105. https://doi.org/10.1016/j.jim.2018.06.023. 

(5) ThermoScientific PierceChicken IgY Purification Kit manual https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011404_Pierce_Chicken_IgY_Purifi_UG.pdf

(6) Sudjarwo, S. A., Eraiko, K., Sudjarwo, G. W., & Koerniasari, Journal of advanced pharmaceutical technology & research. 2017, 8(3), 91-96. https://doi.org/10.4103/japtr.JAPTR_167_16 

(7) Pauly, Diana, et al. Journal of Visualized Experiments. 2011, www.ncbi.nlm.nih.gov/pmc/articles/PMC3197133/.    

(8) Egg Cooking Tips: Cooking With Eggs: Egg Farmers Of Alberta. eggs.ab.ca/eggs/egg-cooking-tips/. (Accessed November 24, 2021)

(9) Constantin, C.; Neagu, M.; Diana Supeanu, T.; Chiurciu, V.; A. Spandidos, D, Exp Ther Med, (2020), 20, 151–158. https://doi.org/10.3892/etm.2020.8704. 
(10) Lu, Y.; Wang, Y.; Zhang, Z.; Huang, J.; Yao, M.; Huang, G.; Ge, Y.; Zhang, P.; Huang, H.; Wang, Y.; Li, H.; Wang, W, Journal of Immunology Research (2020). https://doi.org/10.1155/2020/9465398.

Combatting E. Coli through Secretion Inhibitors

by Akshay Sethi

Introduction:

Escherichia coli (E. coli) is a rod-like anaerobic bacterium that infects the gastrointestinal tract of humans (Fig. 1).

Fig 1: (E. coli bacterial cells)

Most E. coli are harmless, but there are pathogenic strains such as Shiga toxin-producing E. Coli (STEC) that can cause diarrhea, abdominal cramping, and/or acute renal failure. [1] E. coli is found to colonize the human body hours after childbirth and does not pose harm unless the gastrointestinal barrier is broken or the host is immunocompromised. The microorganism is a threat to humans. Gram-negative bacteria possess cell envelopes and an outer membrane that can protect the inner membrane and its organelles from antimicrobial enzymes and antibiotics (Fig. 2). [2]

Fig 2: (Gram-negative bacteria have more components, including an outer membrane, to protect it)

The combination of a low concentration of E. coli needed to infect the host and its resistance to antibiotics makes the microorganism dangerous. [3]. E. coli has a core genome, or series of DNA strands that are common in all strains, but the bacteria also has a non-shared gene pool that differentiates each strain from anotherThere are at least six types of deadly pathotypes of E. coli., all of which cause enteric disease, or diseases relating to the gastrointestinal tract, and potentially lead to long-term injury. 

E. coli is not highly transmissible, as the risks come from contaminated food such as undercooked meat, unpasteurized milk, dirty water, or contaminated stool. [4] Therefore, the best prevention for the risk of infection is cleanliness such as washing one’s hands, as an individual must ingest the pathogen to contract it. [4] 

Novel therapries and treatments for E. coli have shown limited success. [8] Much of the successful experimentation has come from the process of injecting fluid with secretion inhibitors to stop production of key components of the bacterium. The main system for infecting host cells, T3SS, is the main target of secretion inhibitors and has been the focus of modern research on E. coli. [10]

Discussion:

Harms in E. coli:

 E. coli varies in its threat to humans based on the amount of Shiga toxins it produces. [3] Shiga toxins act as ribotoxins that inhibit protein synthesis, leading to altered gene expression or even apoptosis. This would lead to bloody diarrhea, or worse, the Hemolytic Uremic Syndrome (HUS). [5] HUS is one of the more dangerous outcomes of contracting harmful E. coli, where infected people suffer red blood infection and potential total kidney failure. [5] Global estimates project about 10% of STEC infections lead to HUS. HUS is the most common cause of acute renal failure in children, one of the two high-risk groups to contracting an E. coli infection. [5] The syndrome can also cause neurological failures in 25% of cases, resulting in symptoms such as seizure, coma, or stroke. [5]  

Shiga toxins are found in over 200 strains of E. coli. [7] Shiga toxins found in E. coli are found in the pathogenic serotypes of E. coli as Stx1. [4] A serotype is a specific group of bacteria sharing a common antigen, which is an immunogenic particle that incites an antibody response. Another serotype of E. coli contains  Stx2, which is a more potent Shiga toxin and a part of a different antigen serogroup. A serogroup consists of microorganisms differing in their composition of antigens. [4]  The Stx toxin is comprised of two key subunits—the A subunit and B subunit. The A subunit’s function is to inhibit protein synthesis by damaging the ribosome in an infected cell. [4] The B subunit binds to the globotriaosylceramide (Gb3), which is found in endothelial cells lining cardiac muscles and blood vessels. [4] Among biological substances, Shiga toxins are amongst the most poisonous and are lethal to various animals in small doses. E. coli carrying the Shiga toxin serotypes have been shown to lead to the worst outcomes of contracting the pathogen. Across different species, the Shiga toxins have been found to cause acute renal damage and in some cases, total renal failure. [7] Within population-based observations, along with experimentation on mice and baboons, Shiga toxins have been linked to HUS. [4] 

The pathway for Stx serotypes of E. coli to lead to HUS begins with the consumption of contaminated substances. E. coli rapidly replicates within the host and the Shiga toxins latch onto Gb3 before being enveloped and packaged into a microvesicle. This vesicle is then absorbed into the kidney endothelial cells, where the toxins cause necrosis, eventually leading to acute renal failure (Fig. 3). [6] 

Fig 3: (Stx binding to the GB3 on the cell membrane to take over the ribsomal components of the cell)

Besides the kidney, the brain is the second most affected organ in severe E. coli infection. The Shiga toxins attack neurons within the brain and can cause damage or partial failure to the central nervous system. Central nervous system damage can lead to seizure, shock, or paralysis in some cases. [7] 

Classifications of Shiga toxins go more in-depth than Stx1/Stx2 to distinguish between the potency of the toxin and the binding receptor. [7] Some types of Stx can cause severe loss of body mass, along with renal failure. [7] One common symptom of E. coli infection shared between most classifications is colon damage. Shiga toxins inhibit the function of the large intestine, which in turn causes severe symptoms such as bloody diarrhea. [7]

In the past couple of decades, Shiga toxin-carrying E. coli has been found in animal-to-animal transmission. [7] In an isolated study, STEC infection was found in zoonotic transmission, and a common source was domestic animals, such as house cats and dogs. Other sources included wild animals, but at similar rates. Most commonly found were feral hogs and hyenas. Scientists have looked to treat E. coli because of the long-lasting impact of the infection on the human body, including high blood pressure, heart disease, or kidney problems. [1]

Secretion Inhibitors:

One of the newly discovered methods in treating E. coli is anti-virulence therapy. This therapy takes advantage of the type III secretion system (T3SS) in E. coli that primarily exists to grow and multiply the bacteria within hosts. [8] The T3SS acts as a needle that will “inject” effector proteins into the epithelial cells of the gastrointestinal tract. Effector proteins are biomolecules that can attach to proteins and affect the production and type of product that the protein will create. These proteins function as ligands as well as secretory proteins, attaching mainly to host cells to infect them. The ligands will lower the activation energy to conduct the process of making bacterial cells, and therefore increase the rate of production. [8] The impetus for targeting T3SS by scientific researchers is that it is the main mechanism that both Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) use to infect and populate the host effectively. EHEC is the strain of E. coli that can cause HUS, while EPEC is less severe in bodily harm but is the leading strain for diarrheal deaths. [8] 

The method in which the treatment is proposed to work is unique. Instead of regulating or limiting the growth of new cells, the anti-virulent treatment targets and inhibits a virulence factor. [8] Virulence factors are what cause bacteria to cause disease in eukaryotic, multicellular organisms. [8] These factors will include molecules that will aid bacteria in infecting the host organism’s cells. [12] The factors are categorized as either secretory, membrane associated, or systolic. Systolic factos will lead to the bacteria changing its physiological or physical structure. [12] Secretory virulence factors assist the bacterium in counteracting the host’s immune response by releasing chemicals. [12] 

One inhibitor of the T3SS is salicylidene acyl hydrazide, which has been shown to be effective against EPEC, EHEC, and Salmonella. [9] Although effective, the salicylidene acyl hydrazide was discovered to indiscriminately bind to human proteins, which may negatively affect patient metabolism. [10] It is important to note that the effect of salicylidene acyl hydrazide on T3SS is broadly accurate in preventing the secretion system from functioning. [10] 

One important study explores the ways a secretion system inhibitor, Aurodox, can affect the T3SS function in different bacteria. [8] Aurodox is the novel system that incorporates salicylidene. [8] Although it does not affect the growth of bacteria, Aurodox has been shown to inhibit the secretion of T3SS. The effectiveness of Aurodox on inhibiting the T3SS system depends on the concentration of the Aurodox injected into the in vivo sample. [8] Researchers have found that in key strains of E. coli, Aurodox did not inhibit growth of the bacterium while inhibiting the mechanism of T3S production (Fig. 4). [8 ]

Fig 4: (Aurodox will inhibit T3S secretion, but not the growth of different bacterial strains)

In the study, researchers found that Aurodox has the function of inhibiting EHEC to infect epithelial cells in infected mice. Although results were inconclusive at the cellular level, Aurodox was shown to reduce colon damage in the mice. [8] The expression of T3SS in various proteins involved in DNA-binding and altering were also shown to have been inhibited by Aurodox. 

Conclusion:

These results are promising for the inhibition of T3SS expression in anti-virulent treatment. As noted previously, past antibacterial treatments had induced the SOS response in the EHEC strain of E. coli, which was not the case for the anti-virulent Aurodox. [8] This is significant because the SOS response as a function leads to the release of Shiga toxins. Without the release of the deadly toxins, EHEC infection can be inhibited.

Aurodox as a treatment poses an interesting future for combatting E. coli. While Aurodox can effectively inhibit the secretory function of E. coli through the T3SS system, it does not halt production of the bacterium. This poses a question of whether to accept the cohabitation of EHEC and EPEC strains, among others that are considered harmful in the present. [8] If Aurodox can effectively neutralize the harmful effects of the strains, then there is little reason to eradicate the bacterium from the host. 

Drawbacks:

One drawback to current antibiotic treatments which anti-virulent therapy attempts to solve is the SOS response by E. coli. [8] The anti-virulent therapy can cause minor DNA damage, signaling an SOS response. The SOS response will occur after the use of antibiotics and eventually lead to the overproduction of Stx in the intestines. This uptick in Stx production will lead to a higher chance of severe symptoms of the anti-virulence treatment. 

Antibiotic drawbacks are more feared by the scientific community because E. coli is the most common pathogen in humans. In a study analyzing 150 different food samples to test the antibiotic sensitivity pattern of the bacteria, the highest percentage of drug-resistant E. coli was found in the most common foods with E. coli present, which included raw meat, eggs, and salad. [11] To combat this, scientists propose that the general population practice good hygiene, and farmers should only use reserve antimicrobial drugs to reduce the possibility for antimicrobial resistance. [11] Anti-virulence treatment does not deal with antimicrobial drugs, and is more likely to reduce unwanted side effects of general treatment of E. coli as compared to other proposed solutions. 

[1] E. coli. (2018). Retrieved 20 December 2021, from https://www.who.int/news-room/fact-sheets/detail/e-coli

[2] Kaper, J., Nataro, J., & Mobley, H. (2004). Pathogenic Escherichia coli. Nature Reviews Microbiology, 2(2), 123-140. doi: 10.1038/nrmicro818

[3] Fact Sheet: Escherichia coli - Microbial Identification - MALDI ToF. (2021). Retrieved 20 December 2021, from https://wickhamlabs.co.uk/technical-resource-centre/fact-sheet-escherichia-coli/

[4] Melton-Celsa, A. (2014). Shiga Toxin (Stx) Classification, Structure, and Function. Microbiology Spectrum, 2(4). doi: 10.1128/microbiolspec.ehec-0024-2013

[5] Gram-negative Bacteria Infections in Healthcare Settings | HAI | CDC. (2021). Retrieved 20 December 2021, from https://www.cdc.gov/hai/organisms/gram-negative-bacteria.html

[6] E. coli: What is It, How Does it Cause Infection, Symptoms & Causes. (2021). Retrieved 20 December 2021, from https://my.clevelandclinic.org/health/diseases/16638-e-coli-infection

[7] Kim, J., Lee, M., & Kim, J. (2020). Recent Updates on Outbreaks of Shiga Toxin-Producing Escherichia coli and Its Potential Reservoirs. Frontiers In Cellular And Infection Microbiology, 10. doi: 10.3389/fcimb.2020.00273

[8] https://doi.org/10.1128/IAI.00595-18

[9] Zambelloni R, Marquez R, Roe AJ. 2015. Development of antivirulence compounds: a biochemical review. Chem Biol Drug Des 85:43–55.

[10] Dai Wang, Caroline E. Zetterström, Mads Gabrielsen, Katherine S.H. Beckham, Jai J. Tree, Sarah E. Macdonald, Olwyn Byron, Tim J. Mitchell, David L. Gally, Pawel Herzyk, Arvind Mahajan, Hanna Uvell, Richard Burchmore, Brian O. Smith, Mikael Elofsson, Andrew J. Roe, Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds*, Journal of Biological Chemistry, Volume 286, Issue 34, 2011, Pages 29922-29931, ISSN 0021-9258, https://doi.org/10.1074/jbc.M111.233858. (https://www.sciencedirect.com/science/article/pii/S0021925819760700)

[11] Rasheed MU, Thajuddin N, Ahamed P, Teklemariam Z, Jamil K. Antimicrobial drug resistance in strains of Escherichia coli isolated from food sources. Rev Inst Med Trop Sao Paulo. 2014;56(4):341-346. doi:10.1590/s0036-46652014000400012

[12] Sharma AK, Dhasmana N, Dubey N, et al. Bacterial Virulence Factors: Secreted for Survival. Indian J Microbiol. 2017;57(1):1-10. doi:10.1007/s12088-016-0625-1

The Cardiovascular Complications of COVID-19

by William Zhang

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic known as the coronavirus disease 2019 (COVID-19). While initially thought to be a pulmonary virus, scholars have recently argued that the virus may primarily affect the cardiovascular system instead. [1, 2] As such, SARS-CoV-2 remains a pathological mystery for the most part. This paper aims to review the interactions between SARS-CoV-2 and the cardiovascular system as well as how potential SARS-CoV-2 treatments can affect pre-existing cardiovascular complications by looking at the results of published studies. One specific study, published by Shi et al. in the March 2020 issue of JAMA Cardiology, retrospectively assessed the cardiovascular characteristics of 416 COVID-19 patients at the Renmin Hospital of Wuhan University. It was found that approximately one-fifth of the study cohort suffered from COVID-related cardiac injuries. Those who suffered cardiac injuries were of older age, and were associated with increased mortality, necessity for mechanical ventilation, and more severe complications such as acute respiratory distress syndrome (ARDS). [3] On a cellular level, COVID-19 causes cardiac damage through its affinity to the ACE2 protein that is highly expressed in cardiomyocytes. [3,4] Ultimately, a stronger understanding of COVID-19’s cardiovascular pathology is essential for the development of more effective countermeasures and treatments as well as a more specific identification of COVID-19 risk groups. 

Introduction

Since the reporting of its first case in Wuhan, China in December 2019, the novel coronavirus COVID-19 caused over a million confirmed deaths globally. The coronavirus outbreak was declared a pandemic by the World Health Organization on March 11, 2020 and continues to spread rapidly throughout the world with over 122 million confirmed cases as of March 19, 2021. [5] On a macroscopic level, the COVID-19 pandemic has caused global economic downturns due to businesses temporarily shutting down to comply with quarantine. For the average person, the COVID-19 pandemic not only poses physiological risks due to its high mortality rate, but also psychological dangers that can arise through prolonged quarantine and concern for loved ones that may be part of the COVID risk group. As such, an increased pathological understanding of COVID-19 is essential for the development of stronger preventative as well as curative approaches in order for our daily lives to return to normalcy.

COVID-19 is a member of a broader category of viruses known as coronaviruses, which are RNA viruses coated in layers of proteins that typically infect avians and mammals. According to the World Health Organization, COVID-19 is primarily spread through the respiratory tract. When an infected person expels viral droplets through coughing or sneezing, the virus remains in the air. When the airborne virus comes into contact with open orifices such as the eyes, mouth, or nose, it can enter an uninfected person and initiate a new viral infection. [6] While self-diagnosis is possible through detection of critical symptoms such as bluish lips and an inability to stay awake, confirmative diagnosis is performed through real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests on nasopharyngeal swab specimens to detect viral RNA fragments. [7]

Due to COVID-19’s extensive interactions with the human respiratory system, the American Lung Association has classified it as a primarily pulmonary disease. [8] However, the pathology of COVID-19 remains mostly unknown and needs further research. In a recently published study by Reynolds et al., it was found that some COVID-19 patients exhibit hypoxemia due to abnormal vasodilation leading to ventilation-perfusion mismatches, which differs from acute respiratory distress syndrome-induced hypoxemia, where patients receive less oxygenated blood due to alveolar collapse. [9] Researchers began to suspect cardiovascular involvement because the primary receptor for COVID-19 viral entry, angiotensin-converting enzyme 2 (ACE2), is heavily expressed in cardiomyocytes, which renders them susceptible to viral infection and cellular damage. [4] Existing clinical studies also support this approach. One study, conducted by Klok et al., shows that 31% of the 184 Dutch ICU patients enrolled in the study suffered from thromboembolic complications such as ischemic stroke and deep-vein thrombosis. [10] This suggests that COVID-19 has significant cardiovascular effects, and a deeper understanding of its cardiovascular pathology can be groundbreaking for advancing COVID-19 countermeasures.

This paper focuses on the study published by Shi et al. in JAMA Cardiology in order to demonstrate how COVID-19 damages the cardiovascular system as well as how it exacerbates the patients’ pre-existing comorbidities. In this study, approximately 82 of the 416 enrolled patients suffered from cardiovascular complications. Patients with cardiac injury had higher mortality rates (51.2% vs. 4.2%), more need for mechanical ventilation (noninvasive 46.3% vs. 3.9%, invasive 22.0% vs. 4.2%), but also higher median age (74 vs. 60). [3] From these data, it can be seen that the cardiovascular factors that affect a COVID-19 patient’s health are complex, which warrants further investigation. With a stronger understanding of COVID-19’s cardiovascular complications, healthcare professionals can treat risk groups more effectively and therefore lessen the severity of COVID-19 infections in demographics such as elders and patients with pre-existing medical conditions. In addition, we can also better grasp the long-term effects of COVID-19 infections and more readily prepare against them.

Methods

The following methods are paraphrased and taken from the study published by Shi et al. in JAMA Cardiology. [3]

Participants

The study was conducted with COVID-19 patients admitted to the Renmin Hospital in Wuhan, China between January 20, 2020 and February 10, 2020. These patients were diagnosed with COVID-19 with the guidance of the World Health Organization. For the purposes of this study, the enrolled patients who did not present cardiac damage biomarkers such as high-sensitivity troponin I (hs-TNI) and creatine kinase (CK-MB), which are substances that are released into the bloodstream when the heart is under stress, were excluded from the study.

Data Collection

Metrics relevant to the study were collected by researchers from electronic medical records. The data includes: demographics, clinical history, lab test results, and results from cardiac investigations (biomarkers and EKG). Cardiac damage biomarkers were measured upon patient admission, while radiologic data were collected through chest radiography and CT scans. Patients were divided into two groups, one with cardiac injury and another without cardiac injury, where cardiac injury is defined as the expression of hs-TNI above the 99th-percentile upper reference limit. The clinical outcomes of the patients were monitored until February 15, 2020, which was the final date of the follow-up.

COVID-19 diagnoses were confirmed with the Viral Nucleic Acid Kit (Health), which extracted viral nucleic acids. The nucleic acids were then subjected to the 2019-nCoV kit (Bioperfectus), which tests for the N gene and the ORF1ab gene via RT-PCR. Positivity in both tests determine a successful diagnosis of COVID-19.

Statistical Analysis

Categorical variables involved in the study were compared to each other using the Fisher exact test or the χ2 test, while continuous variables were compared to each other using the t-test or the Mann-Whitney U-test. Continuous data were expressed as means (with standard deviation) or medians (with interquartile range), and categorical data were presented as proportions. Survival data were presented as Kaplan-Meier curves, and the survival of patients with cardiac injury versus patients without cardiac injury were analyzed through the log-rank test. Multivariate Cox regression models were used to determine the independent risk factors for death during hospitalization. For all the statistical analyses, P < .05 was considered significant.

Results

The data, figures, and results as presented in this section are all paraphrased and taken from the work of Shi et al. as published in JAMA Cardiology. [3]

Table 1. Baseline Characteristics and Laboratory and Radiographic Findings of 416 Patients With COVID-19. [3]

The retrospective chart study conducted by Shi et al. yielded a large set of patient data demonstrating the interactions between COVID-19 and the cardiovascular system (Table 1). Shi et al. separated the patients into two categories: with cardiac damage and without cardiac damage, and the two patient groups are compared against each other. For this study, cardiac injury was defined as the presence of the cardiac biomarker, hs-TNI, above the 99th percentile.

Statistical analyses show a significantly higher median age for the former group (74 vs. 60, p < 0.001), which implies stronger vulnerability for the elderly against the cardiovascular effects of COVID-19. The statistics for signs and symptoms upon admission are similar between the two groups, but it should be noted that patients who present with chest pain, a common sign of cardiovascular distress, are much more likely to experience cardiac damage from COVID-19 (13.4% vs. 0.9%, p < 0.001). 

Patients with COVID-19 comorbidities appear to have a higher risk of suffering cardiac damage. For example, a higher proportion of patients with cardiac damage suffer from hypertension as opposed to those that are infected by COVID-19 but did not experience cardiovascular symptoms (59.8% vs. 23.4%, p < 0.001). This phenomenon seems to remain consistent in this patient population across most other severe comorbidities known to medical professionals with the exception of pregnancy (0% vs. 2.1%).

Figure 1. Kaplan-Meier survival curves for COVID-19 patients. Mortality over time for patients are graphed from A. time of symptom onset and B. time of admission. In B, the maximum number was 16 days for the population with cardiac injury. C. A comparison of outcomes between patients with and without cardiac injury through log-rank test both starting from time of symptom onset and time of admission.

After analyzing the patients’ pre-admission statistics, Shi et al. also studied the progression of COVID-19 in the two patient populations by plotting their mortality against time (Fig. 1). Patients who suffered from cardiovascular damage from COVID-19 were seen as more severe cases, as it had taken them significantly shorter time to go from symptom onset to follow-up (mean, 15.6 [range 1-37] days vs. 16.9 [range 3-37] days, p < 0.001) as well as admission to follow-up (mean, 6.3 [range 1-16] days vs. 7.8 [range 1-23] days, p < 0.001). Mortality rate was also higher among the patients that experienced cardiac injury as opposed to the non-cardiac injury group (51.2% vs. 4.5%, p < 0.001). 

Table 2. Multivariate Cox Regression Analysis on the Risk Factors Associated With Mortality in Patients With COVID-19

In order to further determine the risk behind cardiovascular damage behind COVID-19, Shi et al. performed a Cox regression analysis on various risk factors and their impact on patient mortality (Table 2). The Cox regression analysis is a model that determines the the effect of multiple variables on a given event through the hazard ratio, and as seen from Shi et al.’s data, the leading risk factor of COVID-19 mortality is ARDS with an average hazard ratio of 7.89 (p < 0.001), followed by cardiac injury with an average hazard ratio of 4.26 (p < 0.001). 

Discussion

Shi et al.’s work shows that despite COVID-19’s nature as a primarily pulmonary disease, its cardiovascular complications are severe and cannot be overlooked. Even though ARDS overshadows cardiac injury as the primary risk factor for COVID-19 mortality, Shi et al. reports that patients with cardiac injury are more likely to need advanced intervention such as noninvasive and invasive mechanical ventilation. [3] Other researchers’ works have agreed with these observations, bringing the cardiovascular complications of COVID-19 to the attention of emergency care workers and even discussing the potential of chronic cardiovascular damage. [2, 11] Zheng et al. have also noted the complex interactions between COVID-19 antivirals and the cardiovascular system, citing “cardiac insufficiency, arrhythmia” among other forms of antiviral-induced cardiac toxicity as a cause for concern for patients with pre-existing cardiovascular complications. [11] While much of COVID-19’s pathophysiology remains unexplored, current research is bringing light to the importance of long-term care for COVID-19 patients even after discharge as well as the necessity for more effective treatment plans that address the severity of cardiovascular damages.

The main observation from Shi et al.’s publication is the association between cardiovascular damage from COVID-19 and its risk of mortality; two risks correlated with COVID-related cardiac injuries are comorbidities and old age. Shi et al.’s findings bring a new understanding as to how the elderly are a risk group beyond their possession of a generally weaker immune system that renders them more vulnerable to viral infections. [12] As the human body ages, so does its organs, and a weaker heart is more likely to be exploited by COVID-19 specifically. While the precise interaction between COVID-19 comorbidities and the virus itself remains unclear, Shi et al. has also cemented a strong association between the two through a cardiovascular perspective. Alongside other present research such as Klok et al.’s study on thromboembolic crises in COVID-19 patients, [10] the results highlighted in this paper will encourage further research to be done on the cardiovascular pathophysiology of COVID-19 in order to better serve known risk groups beyond knowing that the elderly and those with underlying illnesses are more susceptible to severe COVID-19 symptoms.

However, it must be noted that Shi et al. acknowledged the limitations of the study. The ongoing nature of the clinical observations may lead to further conclusions being drawn in the future, and larger patient populations must be observed in order to draw more general conclusions. [3] While the work of Shi et al. supports theories that COVID-19 can directly damage the heart due to its affinity for ACE2, [13] there have also been studies that disagree with the potential for COVID-19 to directly damage the heart. For instance, a study by Xu et al. shows that signs of cellular inflammation have been found in COVID-19 patients without significant cardiac injury, and Shi et al. cited Xu et al.’s work as a potential indication of COVID-19’s indirect involvement in cardiac injuries. [3, 14] As such, the cardiac pathophysiology of COVID-19 remains a mystery, but is certainly an aspect of the disease that necessitates further research. 

Conclusion

Shi et al.’s study was conducted in order to gain a better understanding of the pathophysiology of COVID-19 after observing potential cardiovascular correlations in the patient body. Through clinical observations as well as retrospective chart studies, Shi et al. have found that not only does COVID-19 worsen with pre-existing cardiovascular comorbidities, the presentation of new cardiac injury in COVID-19 patients is strongly associated with mortality. Despite the virus’s tendency to primarily attack the pulmonary system, Shi et al. has shown that the cardiovascular system is also a risk factor to consider, as patients with cardiovascular symptoms typically need more intensive care and intervention. These results are in agreement with existing studies showing that COVID-19 cell entry is dependent on the ACE2 protein that is heavily expressed in cardiomyocytes, which implies that cardiomyocytes are at great risk of being a target for COVID-19. Overall, Shi et al.’s findings suggest that the human heart is an important subject of study in COVID-19 pathophysiology due to its association with increased severity of symptoms. As research progresses, cardiovascular breakthroughs can help with the treatment and control of COVID-19 in the long term. 

Works Cited

[1] Kavanaugh, K. Is COVID-19 Primarily a Heart and Vascular Disease? Infection Control Today, Sep. 8, 2020, https://www.infectioncontroltoday.com/view/is-covid-19-primarily-a-heart-and-vascular-diseases

[2] Long, B et al. Am. J. Emerg. Med. 2020, 38, 1504-1507

[3] Shi, S et al. JAMA Cardiol. 2020, 5, 802-810

[4] Pérez-Bermejo, JA et al. bioRxiv. [Online] 2020. https://www.biorxiv.org/content/10.1101/2020.08.25.265561v1.full. (Accessed October 31st, 2020)

[5] COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University. https://www.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. (Accessed October 31st, 2020)

[6]: Coronavirus disease (COVID-19): How is it transmitted? https://www.who.int/news-room/q-a-detail/coronavirus-disease-covid-19-how-is-it-transmitted. (Accessed October 31st, 2020)

[7]: Country & Technical Guidance - Coronavirus disease (COVID-19). https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance-publications. (Accessed October 31st, 2020)

[8]: Coronavirus Disease (COVID-19). https://www.lung.org/lung-health-diseases/lung-disease-lookup/covid-19#:~:text=COVID%2D19%20is%20a%20lung,other%20than%20supportive%20care%20available. (Accessed October 31st, 2020)

[9]: Reynolds, AS et al. Am. J. Respir. Crit. Care Med. 2020, 202, 1037-1039

[10]: Klok, FA et al. Thromb. Res. 2020, 191, 145-147

[11]: Zheng, Y et al. Nat. Rev. Cardiol. 2020, 17, 259-260

[12]: Meng, H et al. Psychiatry Res. 2020, 289, 112983

[13]: South, AM. et al. Am. J. Physiol. Heart Circ. Physiol. 2020, 318, H1084-H1090 

[14]: Xu, Z et al.Lancet Respir. Med. 2020, 8, 420-422

Epigenetic and Metabolic Shifts in Immune Tolerance

by Daanish Sheikh

INTRODUCTION

Although it is well established that the adaptive immune system can mount an improved immune response upon reinfection with most pathogens, newer insight into innate immune system components suggest that macrophages can be “trained” to also elicit a stronger second immune response, known as immune training. However, the opposite effect, a reduced immune response, has also been observed when monocytes are pre-stimulated with bacterial cell wall component lipopolysaccharide (LPS) [1]; this phenomenon has been termed immune tolerance. Much like how exhausted CD8+ T cells following chronic infections are unable to produce cytokines at optimal levels, tolerized myeloid cells seem to produce fewer cytokines and have diminished phagocytic activity. Monocyte derived macrophage impairment characteristic of immune tolerance has also been linked with immune paralysis following sepsis [2] and in patients recovering from tuberculosis (TB) infection [3-4]. Both TB and sepsis survivors have an increased risk of later secondary infection and an increased risk of mortality [5-6]. Long lasting epigenetic changes are presumed to be responsible for the immune suppression that is observed in these patients [7]. These epigenetic changes are induced by metabolic shifts that upregulate glycolysis, the citric acid cycle, and oxidative phosphorylation, which in turn are activated via the cellular metabolism regulating pathways governed by PI3K, Akt, and mTOR. Pathogen-associated molecular patterns (PAMPs) are recognized by macrophage cell surface pattern recognition receptors whose downstream pathways include these metabolism regulating enzymes. The most well characterized PAMP in endotoxin tolerance involves LPS, which activates the macrophage via cell surface Toll-like receptor 4 (TLR4). PAMP-mediated immune activation also induces epigenetic remodeling of various inflammatory response genes and tolerizing genes. This paper will explore the epigenetic and metabolic shifts that are characteristic of immune tolerance or lead to its development, including the metabolic rheostats by which said long-term epigenetic shifts are mediated.

Myeloid Tolerance and TLR4 Pathway Mediation 

We begin with a brief overview of the pathways downstream of TLR4 and then explore how the pathway varies in immune tolerant cells. Resembling conditions present in sepsis, long term exposure to LPS can result in myeloid immune tolerance; in fact, this model remains the most well characterized example. In LPS tolerance models, immune activation is initially induced via TLR4 exposure to LPS and is transiently characterized by increased energy metabolism via the mTOR and NFAT pathways. TLR4 mediated immune activation occurs via two pathways; first, activation initially results in the production of pro-inflammatory cytokines such as TNF, IL-1, IL-12, and CCL3 via a kinase cascade that includes myeloid differentiation factor 88 (MyD88), various interleukin receptor associated kinases (IRAKs 1,2, and 4), TNF receptor associated factor 6 (TRAF6), and transcription factors NF-κB and AP-1. Meanwhile via a second pathway, the initial activation of TLR4 also upregulates the secretion of interferon ß (IFNß) via a pathway including Toll/IL-1 receptor domain-containing adaptor (TRIF), IKKi kinase, TANK-binding kinase (TBK1), and transcription factor IRF3 [8]. IFNß secretion from this second pathway increases expression of interferon-inducible cytokines that are essential in an immune response [9] (Figure 1). Meanwhile, upregulation of the mTOR and NFAT pathways causes shifts in cellular metabolism that emphasize glycolysis, the TCA cycle, and the electron transport chain, allowing greater energy metabolism by the cell to meet the high energy demands of immune activation [10].

Figure 1: Simplified depiction of the TLR4 pathway and two of its downstream transcription factors

Myeloid immune tolerance is characterized by diminished expression of TLR4, decreased interaction of MyD88 and TRIF with TLR4, and diminished NF-κB and AP-1 signaling. As such, proinflammatory cytokine production is significantly reduced [11]. Upregulation of the mTOR energy metabolism pathways during immune activation is catalyzed via association of upstream kinase PI3K with the TLR4; this pathway is fairly well characterized but summarized here for clarity. PI3K, upon activation via association with active TLR4 and MyD88, phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to form PIP3, which in turn associates with phosphate dependent kinase-1 (PDK-1) to activate protein kinase B (PKB or Akt). Phosphorylated Akt activates the mTOR protein resulting in the aforementioned downstream upregulated metabolic effects [12]. However, diminished expression of TLR4 and MyD88 binding in myeloid tolerance terminates this metabolic upregulation, further exacerbating tolerance.

Metabolic and Epigenetic Modification in Myeloid Tolerance

Some of the causes of myeloid immune tolerance can be attributed to pathways that branch off the TLR4 activation pathway. For instance, PI3K, the kinase implicated in upregulating metabolism, is also implicated in pathways that result in production of anti-inflammatory cytokines [13]. p50, a subunit of NF-κB, is essential for the development of immune tolerance by homodimerization rather than the canonical heterodimerization with p65, another subunit [14]. Other transcription factors that are commonly observed in resting macrophages can also be characteristic of tolerized macrophages. Transcriptional repressor Bcl-6 inhibits the production of inflammatory gene products by regulating the TLR4 transcriptome via association with transcription factor Pu.1. Pu.1 and NF-κB association with inflammatory gene promoters upregulate transcription of inflammatory genes by conferring a tri-methylation to the 4th lysine residue of the H3 histone protein in chromatin (H3K4me3) near those genes [25]. Because Bcl-6 recruits histone deacetylases and histone demethylases to the same site, the same inflammatory genes are downregulated in its presence [24]. Other prominent transcription factors responsible for myeloid tolerance include RelB (a subunit of NF-κB) and high mobility group box 1 protein (HMGB1). RelB complexes with H3K9 methyltransferase G9a to form heterochromatin at the IL-1ß promoter, inhibiting the transcription of IL-1ß, while HMGB1 binds to the TNF-α promoter and recruits RelB complex assembly to downregulate TNF-α transcription [26-27]. It seems clear that transcription factor regulators of myeloid cell activity are highly varied and impact individual gene products allowing a significantly greater degree of control over specific effects of myeloid tolerance as opposed to the widely ranging effects of regulating the TLR4 pathway upstream.

However, over a longer period of stimulation, metabolic shifts in NFAT and mTOR also induce the epigenetic changes that eventually lead to immune tolerance [15-16]. There are three primary links between these metabolic and epigenetic aspects of the cell: Sirtuin1 (Sirt1), alpha-ketoglutarate dependent dioxygenases (αKG-DD), and NuRD10. Activation of Sirt1 occurs at increased concentrations of NAD+, and high dosage LPS exposure drives myeloid cells to upregulate a de novo NAD+ synthesis pathway [17]. Sirt1 is part of the NAD-dependent deacetylase sirtuin family and silences several proinflammatory genes including Tnf and Il1b. Sirt1 also deacetylates RELA, the gene that codes for NF-κB subunit p65 while also binding to RelB, another subunit [18-20]. This SIRT1-RelB complex then serves to assemble a repressor complex that via chromatin methylation later results in endotoxin tolerance18. The second rheostat αKG-DD is a group of epigenetic enzymes whose activity is balanced by αKG and succinate levels in the cell; succinate, as well as various other TCA metabolites including malate, itaconate, fumarate, and 2-hydroxyglutarate, inhibit the activity of these enzymes [21-22]. However, further elucidation of the timing, duration, and relevant metabolites of said TCA cycle shifts to induce epigenetic remodeling by these enzymes is needed. Finally, the NuRD complex is responsible for histone deacetylation and DNA hypermethylation that prevent T-cells from targeting native cells or macrophages from excess inflammation [23]. Upon immune activation, large quantities of reactive oxygen species (ROS) are produced via the electron transport chain; mice with cancer induced immune exhaustion have large quantities of ROS and similar epigenetic changes as to what is induced by NuRD. High levels of mitochondrial ROS also seem to be associated with CD8+ T cell immune exhaustion during TB infection which leads to immune activation via the same Toll-like receptors as LPS [20].

Figure 2: Epigenetic rheostats that link metabolic shifts in the Krebs cycle to immune suppression

CONCLUSION

Although immune tolerance acts as a suppressive mechanism to prevent an excessive inflammatory response, immune tolerance increases the likelihood of secondary infections months following the initial immune challenge; as observed by the long-term morbidity and mortality following chronic infections such as TB and sepsis. Further research considering the possible rescue of immune tolerance phenotypes when cells are exposed to drugs modulating the three aforementioned rheostats should be undertaken to explore the clinical possibilities of reversing immune tolerance and thereby restoring optimal immune function after chronic infection. 

BIBLIOGRAPHY

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28.Images created with Biorender

Impact of Ca(OH)2 and Removal of Egg Shell on pH and Gelation of Chicken and Duck Eggs

Radha Maholtra(1), Michelle Miao(1), Manshi Patel(1), Julia Shi(1), and Caroline McNeil(2)

1 - Rice University Wiess School of Natural Sciences, contributed equally to this work

2 - Rice University Department of Chemistry

ABSTRACT

Using current methods, the process of making century eggs takes roughly 30 days and causes eggs to completely gel. UB Preserv, a Houston restaurant, wants to develop a faster way to prepare century eggs. Two separate experiments were carried out in order to study the impact of the two variables on speed of century egg preparation. First, the concentration of Ca(OH)2 in the egg pickling solution was increased to study its impact on egg gelation speed, and second, egg shells were dissolved to study the impact of increase in the diffusion rate of the pickling solution into the egg. The Ca(OH)2 experiment was carried out using chicken eggs, and the shell experiment used duck eggs. Both experiments recorded a negative result. For the first experiment, the control eggs had a higher internal pH than the experimental. Additionally, none of the chicken eggs completely gelled. All of the eggs became noticeably more viscous over time. For the second experiment, the duck eggs with shells had higher internal pH’s than the eggs without shells. The eggs without shells were significantly stained by the pickling solution and the yolks solidified, preventing yolk pH from being taken. Both duck eggs with shell and duck eggs without shell became more viscous over time, and one control duck egg achieved complete gelation.

INTRODUCTION

Century eggs, also known as pi dan, are a traditional Chinese delicacy. It is made by soaking eggs (traditionally duck) in a mixture of alkaline salt and then fermenting the eggs for a lengthy period while wrapped in solid material which prevents oxygen flow. Through the process, the yolk becomes green while the white becomes dark brown[1]. The transforming agent in an alkaline salt, which raises the pH of the egg to around 10 by the end of the process[2].

Century eggs are part of a specialty dish served at UB Preserv, a Houston-based restaurant. Due to issues with the consistency of commercially sourced century eggs, UB Preserv is seeking a way to make century eggs in house. Unfortunately, the process is much too long for UB Preserv to realistically make this delicacy. UB Preserv reached out to us to see if we could find a way to reduce the length of the egg preparation process.

Previous literature has defined the century egg making process, determined the process raises the final internal pH of eggs[2], and suggested that various cations may impact the speed of egg gelation [3]. Given a study by Ganasen et. al which suggested that Ca2+ ions could decrease egg gelation time, we hypothesized that adding Ca(OH)2 to supplement the alkaline base could speed up the century egg preparation process. In a separate experiment, we dissolved egg shells to increase diffusion rate of the solution across the egg membrane. We hypothesize that this will decrease the time needed for egg gelation by promoting movement of base from the solution into the egg. This movement is what leads to the increase in internal egg pH[1].  The aim in both experimental setups will be to achieve both an increase in internal pH of the eggs and gelation.

MATERIALS AND METHODS

Duck Eggs

Four liters of pickling solution (.62M NaCl, .53M NaOH, 10g/L jasmine tea leaves, milli-Q H2O) were prepared. Shell-less duck eggs—which had been soaked in household vinegar for four days—and shelled duck eggs were rinsed using milli-Q H2O, dried, then placed in the prepared solutions at room temperature.

After soaking for one day, two eggs were removed from each solution, dried, then wrapped in saran wrap to ferment for two weeks at room temperature. This process was repeated every 2-4 days until all the eggs had been removed. After fermentation, the eggs were weighed before separating the yolks and whites. The pH of each component was measured.

Chicken Eggs

Four 1000mL solutions of varying amounts of Ca(OH)2 were prepared (.62M NaCl, .53M NaOH, 10g/1L jasmine tea leaves, milli-Q H2O). Forty-eight chicken eggs were massed and twelve placed in each solution at room temperature.
After 3 days, two eggs were removed from each solution, dried, wrapped in Saran Wrap, and left to ferment for 2 weeks at room temperature. This was repeated every 2-4 days. After fermentation, the eggs were weighed before separating the yolks and whites. The pH of each component was measured.

RESULTS AND DISCUSSION

Chicken Eggs

Our first experiment focused on chicken eggs in solutions with different concentrations of Ca(OH)2. We soaked eggs in solutions of varying Ca(OH)2 content to test the impact of Ca2+ on egg pH. Based on previous literature, we decided to prepare solutions consisting of NaOH, NaCl, jasmine tea leaves, and  Ca(OH)2 [1]. Control eggs were placed in solution with no Ca(OH)2.

The general trend for the average pH of chicken egg yolks over time for all solutions was an initial decrease followed by a period of increase and then decrease. The control solution recorded the highest pH of the four solutions compared. The largest difference between the control and the remaining three solutions was at 15 days: the control yolks had an average pH of 9.85, 56% times more than the average yolk pH of solution 2 and 52% times more than the average yolk pH of solution 1 and 3. (Figure 1.)

Figure 1.

Average pH of chicken egg yolks over time. Two eggs were removed from each solution every 2 days and the pH of the both yolks was measured and averaged. The solutions had Ca(OH)2 concentrations of .000 mM, 67.5mM, 135mM, and 202mM.

For the pH of egg whites, the trend lines generally remained constant before reaching a peak and dropping off. Once again, the control solution recorded the highest pH of the four solutions compared. There was a notable decrease in pH between Days 11-13 for all solutions except the 135mM solution, followed by a peak point at Day 15 in control and the 202mM solution. Between the solutions themselves, the differences in pH between them are not noteworthy; the graph lines often intersect. (Figure 2.)

Figure 2.

Average pH of chicken egg whites over time. Two eggs were removed from each solution every 2 days and the pH of the both whites was measured and averaged. The solutions had Ca(OH)2 concentrations of .000 mM, 67.5mM, 135mM, and 202mM.

Qualitatively, the chicken eggs became more viscous over time, but the whites and yolks did not change color. Additionally, the egg yolks did not have any noticeable changes in consistency. An outlier in the control solution appeared on Day 13 which displayed both the dark brown egg white and green yolk which are characteristic of century eggs.

We expected that by increasing amounts of Ca(OH)2 in each solution, the diffusion rate across the egg shell would increase, making the eggs more basic. By increasing the egg pH faster, we hoped to produce century eggs in less time as a previous study by Ganasen et. al found century eggs to have an average pH of roughly 10 by the end of the process[2]. However, our results disagreed with our expectations: higher Ca(OH)2 concentrations led to lower pH of the egg yolks and whites. 

This result may be because we were unable to get the Ca(OH)2 to fully dissolve. We believe this to be because the solutions were fully saturated. A potential consequence of this is that the solution surrounding the eggs did not diffuse into the eggs. The saturation of solution could have led to a hypertonic solution when compared with the egg, leading any diffusion to occur from the egg into solution, rather than the other way around. Given these results, we suggest that Ca(OH)2 actually makes the process of achieving egg gelation slower because our experimental eggs recorded lower pHs than our control eggs and because the Ca(OH)2 may have caused the solution to be hypertonic to the egg which would interfere with the diffusion of the solution into the egg.

Duck Eggs

Our second experiment sought to determine whether the absence of the egg shell would increase the diffusion rate of the pickling solution into the eggs. Eighteen experimental duck eggs were soaked in household vinegar to dissolve the duck egg shells without destroying the egg membranes. Eighteen control duck eggs were not placed in vinegar so that their shells were maintained. 

The pHs of the duck egg yolks remained mostly constant throughout the experiment, with the exception of a slight decrease in pH at Day 4 in the control eggs and slight increase in pH on the same day in the experimental eggs. Our control eggs consistently had higher yolk pH than our experimental eggs (Figure 3.).

Figure 3.

Average pH of duck egg yolks determined every 2 days. After Day 6, pH testing of the shell-less eggs ceased due to solidified yolks.

For the average pH of whites, the experimental eggs remained constant throughout the experiment until the end, where there was a sharp increase in pH. The control eggs had a slight increase in pH before dropping at Day 11 and then increasing again. Once again, our control eggs had a higher pH than our experimental eggs. The largest difference could be seen after 8 days in solution, where the pH of control egg whites was pH of 10.65; this was 69% more than the pH of our experimental egg whites, which was 6.3 (Figure 4.).

Figure 4.

Average pH of duck egg whites determined every 2 days. The pH of egg whites without shell was not taken for eggs after Day 13 as there was significant fungal growth on the eggs, which would have introduced a confounding factor into the data.

Qualitatively, the experimental egg yolks became solid at Day 6 while the whites became more viscous over time. The control egg whites and yolks remained largely unchanged. When massed, experimental duck eggs were significantly larger than control duck eggs. Shell-less eggs were also notably more stained outside than were shelled eggs, and the experimental egg yolks became a muddy yellow color while control egg yolks remained bright yellow. An outlier control egg that was in solution for 13 days achieved complete gelation of the egg white.

We expected the removal of the egg shell to lead the eggs to reach higher pH faster. By removing the shell, we remove a barrier between the eggs and solution, increasing diffusion. The movement of base into the egg is what leads to the increase in egg pH, so we hoped that removing the shell would increase the speed of diffusion and thus help the eggs increase pH[1]. However, our results disagreed with our expectations; the eggs without shells had a lower pH than our control eggs. Additionally, the pH of the control egg whites increased sooner than the pH of experimental egg whites, which had mostly constant pH. This may have been due to the vinegar used to dissolve the egg shells. Vinegar contains acetic acid, which has a low pH. This could have impacted the pH of the duck eggs’ yolks and albumen significantly.

Noticeable fungal growth was observed on our experimental eggs on Day 13. The growth was due to humid and warm laboratory conditions which were outside of our control. These conditions promote fungal growth[4]. The pH of the duck eggs without shells could have been influenced by the growth of fungi, which would introduce a confounding variable into our study. Thus, we did not include these final few eggs in our results.

CONCLUSION

We carried out two separate experiments in order to study the impact of Ca(OH)2 and removal of the egg shell on egg pH and speed of gelation. Both experiments recorded negative results; the control egg groups in both experiments had higher internal pH than the experimental egg groups. Additionally, none of the chicken eggs completely gelled, though the eggs became noticeably more viscous over time. These results may be due to undissolved Ca(OH)2 in the pickling solutions in the first experiment and the impact of the egg shell removal process on egg pH in the second experiment.

In future experiments, the process of dissolving the egg shells could be modified so that the shell is instead partially dissolved. This would allow the diffusion rate to increase by removing some of the shell while preventing the duck eggs’ pH from being influenced to the same extent by the vinegar as in our experiment. Furthermore, the chicken eggs could be pickled with metal cations (e.g. Zn2+), which have been suggested to help eggs retain solution[5].  Fermentation time could also be reduced to prevent excess denaturation, which can lead to liquidation of the egg yolk after solidification and fungal growth [4]. Fungal growth specifically would make the eggs inedible, which defeats the purpose of decreasing the time needed to prepare century eggs so that they can be served in a restaurant. Environmental factors such as humidity and temperature should be better controlled in future studies to ensure that the results are consistent and not impacted by external variables such as fungal growth.

ACKNOWLEDGEMENTS

This work was supported by Rice University Wiess College of Natural Sciences. Additional thanks to Dr. Jamie Catanese, Sydney Parks, and Kevin Sun for their guidance and Dr. Lesa Tran Lu for providing the duck eggs utilized in our experiments.

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Fructose: The Sneaky Saccharide

by Vanessa Garlepp

It’s Saturday night. You’ve had a long week, and it’s time to settle down for a quiet dinner at home. Picking up your phone, you call McDonald’s to place an order and soon find yourself enjoying a grilled chicken sandwich with barbecue sauce, plus a side of apple dippers and a fruit and walnut salad. Good work–you’ve managed to craft a healthy meal from a fast food restaurant. Or have you? 

What if you discovered that your dinner contains approximately 29 grams of fructose, [1] a fruit sugar found in most American foods [2]? Fructose is widely integrated into the Western diet due to its sweetness–up to 1.28 times sweeter than table sugar–and inexpensive manufacturing process [3]. The human body can comfortably process up to 25 grams of fructose per day [2]; however, beginning in the late 1900’s, the introduction of high-fructose corn syrup (HFCS) to the Western diet has increased average fructose consumption to 85-100 grams per day [4]. This means that in a single meal, you may have consumed more fructose than your body can comfortably handle. Perhaps it’s time to rethink your McDonald’s order.

U.S. fructose consumption is dominated by sweetened beverages (30.1%), grains (21%), and fruit/juices (19.4%) [5]. Almost every soda contains large amounts of HFCS, leading to exorbitant fructose consumption: for example, a medium Dr. Pepper soft drink contains 27.25 grams of fructose, [6] and one 16-ounce Sprite contains 32.4 grams [7,8].  And yes, fruit: despite its numerous health benefits, fruit is the number one source of naturally occurring fructose. One apple contains about 7.5 grams, and one cup of grapes contains 11.7 grams [6].

It may be simple enough to avoid these obvious sugar sources; unfortunately, many “healthy” foods also contain high amounts of fructose. Despite studies proving that sports drinks with carbohydrates (including fructose) cause gastrointestinal symptoms and poorer overall performance, companies such as Gatorade still add excessive amounts of fructose to their products. For example, one 20-ounce bottle of lemon-lime Gatorade includes 15.5 grams of fructose [6,9]. Granola bars are another common source of fructose: HFCS can be used to hold the ingredients together, resulting in up to 11.1 grams of fructose per bar–sweeter than a full-sized Kit Kat [6]! This tendency of “healthy” foods to conceal high levels of fructose can be extremely detrimental to those who turn to them for nutritional benefits.

As a consequence, fructose constitutes over 10% of Americans’ daily calories [5]. The age group with the highest fructose consumption level is adolescents (ages 12-18), who average a daily fructose intake of 72.8 grams [5]. This is likely a result of the vast amount of junk food available to teenagers from vending machines, fast food restaurants, high school/college cafeterias, etc. However, high-fructose diets are also common among young children–kids between the ages of 2 and 5 consume an average of 44.9 grams of fructose daily, which is almost double the amount of fructose an adult can process [5,2]! The main problem? Fruit juice. Despite the widespread belief that juice is a healthy beverage, almost all “100% fruit juices” contain added sweeteners. For example, a single box of apple Juicy Juice–one of the most common juice brands–houses 8.1 grams of fructose alone [7].

In addition to increasing calorie intake, high-fructose diets lead to severe biological consequences that are manifested throughout the U.S. population. Excess fructose consumption triggers enhanced lipogenesis, a process of fatty acid synthesis which causes the buildup of triglycerides (fat) [4]. Furthermore, as opposed to its counterpart (glucose), fructose fails to stimulate insulin secretion and is not carried to the brain upon absorption [3]. Since insulin is a key hormone that regulates food intake and body weight, its absence leads to increased fat storage [3]. Additionally, since fructose does not reach the brain, it cannot signal satiety and may cause the consumer to continue eating without realizing that he or she is full [3]. This combination of biological mishaps can lead to metabolic syndrome, a cluster of conditions (including increased blood pressure and blood sugar, insulin resistance, and obesity) that increases the risk of cardiovascular disease and Type 2 diabetes [4]. High-fructose diets can also cause lasting detrimental health effects in children: the high fructose contents of “100% fruit juice” caused a doubling of childhood asthma in the years 1980-1995 [11]. Therefore, an increased awareness of the prevalence of fructose in the American population is necessary in order to prevent these serious health concerns. 

Due to the pervasiveness of HFCS in the U.S., fructose consumption may be inevitable; however, there are some strategies you can implement to avoid flooding your system with fructose. First, always read ingredient labels on store-bought items. Even if it seems unlikely that a certain product will contain HFCS, there are several fructose-containing foods that may surprise you: that barbecue sauce that you decided to include with your dinner contains 8.75 grams [1]. In addition, choosing foods that contain equal amounts of glucose and fructose can mitigate the negative effects of fructose on your body [5]. When eaten on its own, fructose is poorly absorbed by the intestine, enters the colon as a complete saccharide, and is fermented there [10]. This causes gastrointestinal distress and excessive gas, and is commonly known as fructose malabsorption [10]. Luckily, fructose absorption is aided by the presence of glucose, so choosing foods containing a fructose:glucose ratio near 1:1 can mitigate digestion issues [5]. Instead of buying the Juicy Juice cited earlier–which contains a F:G ratio of 2.38:1–consider Tropicana 100% Orange juice, which boasts a more reasonable ratio of 1.17:1 [7].

Given the presence of hidden HFCS in everyday foods, avoiding fructose altogether may prove to be an unreasonable goal. However, by reading ingredient labels and choosing to consume fructose in foods with an equal amount of glucose, it is possible to mitigate the negative health consequences of fructose overconsumption. Many health programs seek to increase awareness about the dangers of fructose and reduce its widespread availability, particularly in school cafeterias. Nonetheless, the next time you order McDonald’s on a Saturday night, try swapping out barbecue sauce for ranch dressing or trading the apple dippers for a fruit and yogurt parfait. This way, you can implement your new knowledge of high-fructose foods to make informed decisions and avoid unnecessary fructose consumption. 

[1] Traditional Oven. https://www.traditionaloven.com/foods/menu/fast-food/ (accessed 10 Oct., 2020)

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[8] The Coca-Cola Company. https://www.coca-cola.ca/brands/sprite (accessed 9 Oct., 2020)

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