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Clouded Vision: An Introduction to Cataracts

Cataract timeline

Cataracts have been around since the Axial age, before 500 B.C. The first documented case was a surgery by Indian surgeon Sushruta and have been prevalent in medicine ever since. Cataracts are the number one cause of blindness in the world, so it is obvious why studying them is extremely important1. Cataracts are defined as lens opacities, derived from the fact that they look like a white spot has covered part of the eye, similar to what an egg white might look like. It could be anywhere from a small speck, to covering the whole pupil. 

Figure 1. Healthy vs cataractous lens. Note: this is not the same eye. 

Cataracts affect the lens of the eye. The lens helps focus light onto the retina, which converts light signals to electrical signals so the brain can interpret them. The lens is located directly behind the pupil, so the cataract is visible through the pupil as demonstrated in Figure 1. 

I’m sure most of you readers have heard of cataracts before, known someone with them, or may have had them yourselves! You might even be aware of some of their symptoms. Cataracts are most primarily marked by the presence of the white film as I stated, which can impair the vision in a multitude of ways. They are able to lead to blurry vision and even the complete loss of sight if severe enough2. I’m sure you can imagine that if you have a huge white blob covering your eye that it could heavily impact your ability to see. It is comparable to looking through a fogged up window or dashboard. Cataracts can cause objects to seem dimmer, affect night vision, a sensitivity to light, and double vision. Changing glasses and prescriptions can help some of these symptoms, but once cataracts begin to disturb your daily activities it is usually time to take care of the issue through surgery. Cataracts can be harmless, but typically the problem worsens over time and can cause complete blindness if not treated. Clinicians can identify cataracts through the physical appearance by a routine eye exam where they examine the eye’s structures with a slit lamp2. If you have been to the eye doctor, you may have been in this position where a strong light is shined in your eye and your are told to look at the doctors’ ear/earring to keep your eye focused and open (usually causing you to get a little teary eyed, in my personal experience). This allows them to better see the severity of the white blob rather than just by the naked eye. 

Figure 2. Slit lamp examination and cataract result3. 

The lens is made up of mostly proteins, which help focus the light onto the retina. The most important structural proteins in the eye are called crystallin proteins, which make up 90% of the proteins in the eye4. They have to be tightly packed in order to correctly refract the light onto the retina. Ironically, these proteins do not even form crystals as this would create alternate paths for the light, in fact they do the exact opposite. There are three different types of crystallins with two being purely structural, while the other has the ability to prevent the formation of large clumps of proteins. These clusters of proteins are called aggregates and are very dangerous to the health of the eye. α-, ꞵ-, and 𝛾-crystallins all have the structural component to direct light, while only α-crystallins protect the structure of other proteins in the cell, including the ꞵ- and 𝛾-crystallins5. Crystallin proteins are made at birth, and do not undergo any regeneration so they have to last your whole lifetime! It is easy to see how this can lead to issues with your eyes if these proteins are altered in some way and stop working. 

Cataracts can either occur at birth or a young age, called congenital cataracts, or later in life due to aging. Each type of cataract can have various causes. Congenital cataracts may occur due to medications of infections the mother experienced or be inherited. Inherited cases account for 10-25% of congenital cataracts and cause changes to the genes for crystallin proteins4. This causes them to be unable to direct light correctly or the breakdown of proteins by α-crystallin and cause the buildup of crystallins into the white blob you may see. In cataracts due to age, modifications can be made to the proteins over time which alters their structure and prevents them from performing their job1. Additionally, microstructures of the cell such as vacuoles can change the refraction of light and change the ability to see. Vacuoles are structures of cells which hold liquid, so an increased number of them can alter light scattering and cause visual impairment. This in turn can compromise the health of the cell and lead to precipitation of proteins and worsen the issue. 

Different causes of cataracts additionally form in different areas of the eye – nuclear, cortical, and posterior. Cortical cataracts most often are associated with the cell membrane health, nuclear with the breakdown of proteins, and posterior with medication side effects, trauma, and other eye issues6.

Thank you so much for reading and I encourage you to check out the rest of my blog for further in-depth content about cataracts!

Table of Contents

Crystal Clear: The Function of Crystallin Proteins

Who Let the Cataract Out of the Bag: Discovery and Diagnosis

Mechanism of Disease

I Can See Clearly Now, the Cataracts are Gone: History of Treatment

References

  1. Bloemendal 2004
  2. https://www.mayoclinic.org/diseases-conditions/cataracts/symptoms-causes/syc-20353790
  3. https://www.aao.org/eye-health/diseases/what-are-pediatric-cataracts
  4. Hejtmancik 2008
  5. Hejtmancik 1998
  6. Chylack 1993

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Lipid Biotechnology: the War on Antibiotic Resistance

If you’ve ever been to the doctor and diagnosed with a bacterial infection, you were probably prescribed an antibiotic. From their discovery in the early 1900s, antibiotics were defined as inhibiting the growth of or killing bacteria via a chemical substance (Waksman, 1947).

 The most prevalent antibiotics are B-lactam derivatives which  target penicillin binding proteins, inhibiting peptidoglycan synthesis and resulting in cell lysis. 

Antibiotic resistance’s prevalence came about in the 1930s, beginning with cases of antibiotic resistant syphilis in Western Europe and increased toward the end of the decade (Beckh and Vulgar 1939). Since then, it has become one of the world’s leading public health issues (WHO). This has been due to the incorporation of antibiotics into farming as well as the sanitation conditions in developed countries (NIH). Specifically, the overuse of antibiotics allows the non resistant bacteria to be killed, and the resistant survive and pass on the gene to the next generations. There have been many efforts to efficiently combat this antibiotic resistance: intracellular antibiotic delivery, lipopeptide antibiotic tripopeptin C, and B-lactamase inhibitors (Laws et al, 2019). The specific mechanism of antibiotic resistance occurs via B-lactamase, which hydrolyzes the B-lactam ring by cleaving the amide. 

Recently, antisense therapy has been used as a method of combating antibiotic resistance when these have proved to show poor efficacy (Chan et al., 2006). This constitutes the use of oligonucleotides, which are short sequences of synthetic DNA. They are known as oligonucleotides (ONs) or antisense oligonucleotides (ASOs). They target certain mRNA sequences by binding them via a complementary sequence of a gene.

They can either work within the nucleus preventing 5’ cap formation and telomere addition, or in the cytosol by binding to the Shine-Dalgarno sequence and blocking the ribosome from transcribing the gene. However, the largest issue occurs when the oligonucleotide can not enter into the cell to reach the mRNA due to its large phosphate backbone not being able to cross the membrane. This led to the authors’ decision to attempt lipid conjugation of the molecule (Kauss et al, 2020).

In solution, oligonucleotides remain aqueous and therefore cannot pass through the membrane. Lipid oligonucleotides will form micelles, or an aggregate of the molecule where the hydrophobic parts will surround the outside with the hydrophilic parts facing the inside. This in turn allows the molecule to pass through the hydrophobic core of the lipid bilayer. As demonstrated by Karaki et al, lipid conjugated oligonucleotides enter the cell by endocytosis, and at a much greater rate than non conjugated molecules (2017). They were able to decrease tumor proteins that led to prostate cancer cell characteristics. 

Cephalosporins are one of the major classes of antibiotics, which can be targeted by B-lactamases. Using lipid oligonucleotides to target the B-lactamase mRNA is proposed to restore antibiotic sensitivity by blocking its transcription and enzyme production. 

The authors used a sensitive and resistant laboratory strain as well as another resistant clinical strain to demonstrate their findings. First, they tested the viability of cells in the presence of oligonucleotides and lipid oligonucleotides respectively. The sensitive strain’s reaction to cephalosporins was unaffected by either oligonucleotides. However, both the laboratory and clinical resistant strains had a decreased minimum inhibitory concentration (MIC) with the lipid oligonucleotides, meaning that a lower concentration of antibiotic was able to inhibit the cells. Their control was a scrambled sequence of nucleotides that would not target specific mRNA, and that did not have any effect on the MIC.  

They experimented with structure in a few different ways in order to engineer the best possible molecule. They tagged the lipid onto the 5’ and 3’ ends and found that the 3’ end increased the MIC slightly. This led them to use the 5’ version as it had the greatest efficacy of decreasing MIC. Additionally, they altered the number of base pairs and found that the 19-25 bp range produced the lowest MIC. 

Then, they sought visual proof that the lipid oligonucleotide entered the cell. To visualize intra bacterial localization they tagged LON with Cyanine 5 fluorescent dye and analyzed by fluorescent microscopy. 

In order to track the B-lactamase activity they used a cephalosporin called nitrocefin that changes color from yellow to red when hydrolyzed. They measured the absorption at 492 nm with varied oligonucleotide concentrations. They found that 5 uM lipid oligonucleotide hydrolyzed the least antibiotic, displaying that it was the most effective concentration. However, the figure does not show if more than 5 uM was tested, so we do not know if there was a more effective concentration or if increasing the concentration did not decrease MIC after 5 uM. 

RT-PCR and western blotting on the transcripts and protein products was performed to test expression. Similar levels were present when the nonconjugated versus the lipid oligonucleotide were used, indicating that the lipid conjugated molecule did not affect the production of B-lactamase compared to untreated control. A specific mechanism was not investigated by the authors, but it is probable the oligonucleotide produces some off-target pathway based on the decreased MIC but lack of change in transcript concentration. 

Ultimately the authors were able to demonstrate that lipid oligonucleotides provided increased efficacy of antibiotics by decreasing B-lactamase activity in hydrolyzing B-lactam rings. They designed their molecule through trial and error and deduced that lowering the minimum inhibitory concentration is LON concentration-dependent. This was the first time this was performed in a gram negative species, the lipid micelle able to enter the cell by endocytosis through the peptidoglycan and cell wall layers. This technique of lipid conjugation could lead to discoveries in drug delivery across cell membranes as well as further advancements in combating antibiotic resistance if the mechanism is investigated.

References

Original article: Kauss, T., Arpin, C., Bientz, L. et al. Lipid oligonucleotides as a new strategy for tackling the antibiotic resistance. Sci Rep10, 1054 (2020). https://doi.org/10.1038/s41598-020-58047-x

Waksman, Selman A. “What Is an Antibiotic or an Antibiotic Substance?” Mycologia, vol. 39, no. 5, 1947, pp. 565–569. JSTOR, www.jstor.org/stable/3755196. Accessed 5 Feb. 2020. 

World Health Organization Antibiotic Resistance. https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance

Causes of Antimicrobial (Drug) Resistance. https://www.niaid.nih.gov/research/antimicrobial-resistance-causes

Mark Laws, Ali Shaaban, Khondaker Miraz Rahman, Antibiotic resistance breakers: current approaches and future directions, FEMS Microbiology Reviews, Volume 43, Issue 5, September 2019, Pages 490–516, https://doi.org/10.1093/femsre/fuz014

Chan, J. H. P., Lim, S. & Wong, W. S. F. Antisense oligonucleotides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol. 33, 533–540 (2006).

Karaki, S., Benizri, S., Mejías, R., Baylot, V., Branger, N., Nguyen, T., Vialet, B., Oumzil, K., Barthélémy, P., and Rocchi, P. (2017) Lipid-oligonucleotide conjugates improve cellular uptake and efficiency of TCTP-antisense in castration-resistant prostate cancer. Journal of Controlled Release 258, 1–9.

Beckh W, Kulchar GV. Treatment-Resistant Syphilis: An Evaluation Of The Causative Factors In Eighteen Cases. Arch Derm Syphilol. 1939;40(1):1–12. doi:10.1001/archderm.1939.01490010004001


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Reflection Blog 2

The most basic description of an optometrist is to examine the eyes for defects or abnormalities, whether it be through refractive error or the presence of a disease which may very likely affect vision. The ranges of an optometrist’s ability to treat is from prescribing glasses to eye drops as well as other methods of treatment such as warm compresses. My initial interest in optometry came from the observation of simple eye exams and prescribing glasses to children, while it has now been able to flourish through shadowing into viewing holes in a patient’s retina or the migration of the lacrimal gland to the front of the eye, creating an aesthetically displeasing bulge. This interest drives me to primarily focus on ocular diseases and wanting to discover their biochemical and genetic basis. I am excited to combine both of my main interests of biochemistry and optometry and have them ultimately culminate into one research project.

The first disease I would like to investigate is diabetic retinopathy. Although it is usually characterized as a “microvascular complication of diabetes,” research has been found that actually marks it as first being a cause of neurodegeneration by continuous inflammation of the retina and glia (Sinclair and Schwartz, 2019). The rare ability of these symptoms to be diagnosed early enough severely limits the success of treatments. There are many genetic and physiological factors which play into this disease, some genes being strongly linked to DR but none having extremely concrete evidence.

The next ocular disease I am interested in is color blindness, specifically the red-green variation that is the most common. As taught in any biology class, being color blind follows classic Mendelian genetics via a X-linked recessive disorder. The OPN1LW and OPN1MW genes are responsible for providing a photopigment called an opsin that allows an electrical signal to be sent to the brain when long and medium wavelengths of light are detected, respectively, by the cones (NIH).

Cataracts are defined as ocular opacities, ultimately blocking vision. Those that are age-related account for 50% of blindness worldwide. They arise from a range of genetic mutations including cytoplasmic crystallins, membrane proteins, cytoskeletal proteins, and DNA/RNA-binding proteins with a mostly autosomal dominant pattern. I am most interested in the crystallin mutations which alter folding and solubility (Shiels and Hejtmancik, 2014).

References

OPN1LW gene – Genetics Home Reference – NIH. U.S. National Library of Medicine. National Institutes of Health.

Shiels, A. F., and Hejtmancik, J. F. (2014) Molecular Genetics of Cataract. eLS.

Sinclair, S. H., and Schwartz, S. S. (2019) Diabetic Retinopathy–An Underdiagnosed and Undertreated Inflammatory, Neuro-Vascular Complication of Diabetes. Frontiers in Endocrinology 10.

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Why Biochem?

Well.. my story started when I was a wee highschool student taking AP biology. I found my interest to be rooted in the DNA/genetics portion of the class, rather than the later units focusing on ecology (yuck). I aced the translation and transcription exams in my class, no surprise there! Since then, I knew I was just DESTINED to go into the sciences and a health profession. It wasn’t until I got to college and started out with Bio I, again encountering the dreaded taxonomy and similar topics. I found myself unable to wait to take Bio III, as I heard of a magical class full of DNA, cancer biology, and experimental techniques. I was told that Bio III was the gateway drug for future biochemistry majors, and after loving the class I sought out Dr. Hark to get her opinion and hopefully get my major declaration form signed. At this point I had never heard of the amazing Keri Colabroy and would not meet her until months later, scared out of my mind to meet the infamously intimidating professor. What Dr. Hark had explained to me was that biochemists like to know the why and how of biological processes within the body system, a combination of all different kinds of chemistry and biology. What I soon learned from the biology classes I took was that they were more focused on the “what,” and characterization of animals, their behaviors, and bodily systems. 

When I came to truly start my journey in biochemistry I was opened to a world of metabolic pathways, amino acids, and of course, my personal favorite, enzymes. I initially began biochemistry because I wanted to be able to explain and investigate the pathways and reactions occurring in the body, but starting research made me realize it would really teach me so much more. As I delved into actual experimental biochemistry and beginning my own research project, I soon gained so much knowledge on enzymatic reactions and mutating genes at your will that I could never get enough, leading me to stay for two summers doing research. Being a biochemistry major has given me an advantage in class of looking deeper into the root of the problem and searching for a solution that is not automatically characterizable and might be caused by multiple sources in the body. 

Finishing almost all my biochemistry coursework at this point in time, it astounded me how much all of my classes have related to each other and how interdisciplinary each subfield of biology and the other sciences truly are. Learning of the vibrational and other modes of electrons and atoms in physical chemistry, in my receptors and channels course this was related to the motion of receptors and structures of the membrane – no entity being truly stagnant or staying the same. My physical chemistry course debunked all of the knowledge I thought to be true of entropy and the location of an atom, each year of college building on one another. 

Although not quite related to biochemistry in the traditional sense, my desire to enter into the optometry field has only been heightened from my coursework. All the general courses as well as biochemistry, microbiology, and of course optics from physics will be part of my first year of optometry school and Muhlenberg has prepared me at length for upper level work. My research has pushed me to want to become a clinical professor at some point in my life, and hopefully educate the future of optometry (once I become a part of it). For my project this semester, although I have not thought too extensively on it, I would like to study an ocular disease and hopefully be able to add this work to my CV showcasing my interest and present knowledge of optometry!