The Baldwin Review 2018

The Baldwin Review A collection of individual research papers produced by Upper School students of The Baldwin School

Foreword Two years ago, Eliza Thaler ’18 founded The Baldwin Review with the purpose of capturing the intellectual spirit of Baldwin students in their academic endeavors outside of Baldwin’s gates. We are honored to be continuing her legacy with the publication of the journal’s third edition. This year’s collection includes 10 scientific research papers written by members of the Class of 2019, who spent their summers in labs and hospitals in the Greater Philadelphia area as well as Shanghai, China.

Each paper in this journal is the culmination of months of hard work and dedication.

While reading through each one, we were struck by the sophistication and rigor of each body of work; each exemplifies the curiosity and global impact of Baldwin students. We are grateful to have been a part of The Baldwin Review these past two years and are excited to see Baldwin girls continue to be at the forefront of academic pursuits in the years to come.

- Anoushka Gidh and Simi Bleznak, Class of 2019

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The Baldwin School, an independent college preparatory school, develops talented girls into confident young women with vision, global understanding and the competency to make significant and enduring contributions to the world. The School nurtures our students’ passion for intellectual rigor in academics, creativity in the arts and competition in athletics, forming women capable of leading their generation while living balanced lives.

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Table of Contents A1

SIMI BLEZNAK ’19 Analysis of Anti-MOG Data For Pediatric ADEM Patients

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ANOUSHKA GIDH ’19 Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications

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ANISHA DEVAS ’19 Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors

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ALEXA DIECIDUE ’19 Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer

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MERIEL DOYLE ’19 Construction of a Chimeric Adeno-associated Virus 2/Human Parvovirus 4 Vector for Gene Therapy

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HELEN JI ’19 Comparison of immunofluorescence signal amplification of a commercially available kit with other amplification techniques

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SHIYU LI ’19 Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells

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MINGXIN SHI ’19 Preparation of Porous and Recyclable PVA-TiO2 Hybrid Hydrogel

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ANJALI SUKHAVASI ’19 Decreasing Pancreatic Ductal Adenocarcinoma Cell Viability by Increased ROS Levels Through IDH1 Inhibition

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TAYLOR TRAPP ’19 Role of Mitochondrial Haplogroup Variants Determine Mitochondrial Dysfunction and Clinical Outcome in Pediatric Sepsis

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SIMI BLEZNAK ’19 Simi Bleznak, a senior from Wynnewood, PA, has attended Baldwin since Pre-K. She is Co-Editor in Chief of The Baldwin Review, Co-Head of Lamplighters (Baldwin’s student tour guides) and Co-Head of Model Congress. In addition, she is the captain of the soccer and basketball teams. Simi enjoys watching football and taking pictures in her free time.

Analysis of Anti-MOG Data For Pediatric ADEM Patients

By Simi Bleznak ’19

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

Acute Disseminated Encephalomyelitis (ADEM) is a short but acute inflammatory attack in the brain and spinal cord region. Like Multiple Sclerosis (MS), ADEM is a demyelinating disorder, one that damages the myelin sheaths in the neurons. Unlike MS, however, it is more commonly diagnosed in children and more specifically males.1 Myelin Oligodendrocyte Glycoprotein (MOG) is an adhesion molecule expressed in the myelin sheaths in the nerves in the Central Nervous System (CNS). Some people, however, have an antibody to this protein. Recent findings suggest that ADEM is often a clinical presentation of anti-MOG antibody positive patients. Through analysis of data on pediatric ADEM patients, the phenotypes of those with and without antibodies to the MOG protein are trying to be characterized.

In total, there were 68 patients in the ADEM cohort; 31 patients were determined to be anti-MOG antibody positive and 31 anti-MOG antibody negative. Six were labeled as anti-MOG antibody “low positive” based on the concentration of the antibody present. For that reason, they have been included in this chart but excluded from the rest of the analysis. The number of female and male patients in the positive and negative groups were nearly equal; however, the anti-MOG antibody positive cohort had a median age of 4.95 years compared to 7.01 years in the negative group, a notable difference.

“Acute Disseminated Encephalomyelitis (ADEM)," National Multiple Sclerosis Society, accessed September 18, 2018, https://www.nationalmssociety.org/What-is-MS/Related-Conditions/Acute-Disseminated-Encephalomyelitis(ADEM).

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

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Follow-up appointments occurred at the following time points: three months after the initial visit, six months after the initial visit, one year after the initital visit, and anually after that. The chart above depicts the number of patients present at the respective follow-up visits, with the green representing the anti-MOG antibody positive patients and the red representing anti-MOG antibody negative patients. At the initial appointment, there were 31 patients present in both groups. However, at each successive visit, fewer anti-MOG antibody positive patients were present.

While some patients were persistently positive or negative for the antibody across all visits, others were not. At the three month follow-up, four patients who were initially anti-MOG antibody positive became anti-MOG antibody negative. At the six month visit, one more previously antiMOG antibody positive patient became negative. There was one outlier patient who, at the three month visit, was determined to be anti-MOG antibody positive after being negative at onset.

Although the number of patients present at each visit declined, five patients were present eight years after their initial appointment.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

Another depiction of data expressed in the demographics chart, 55% (17) of anti-MOG antibody positive patients were female and 45% (14) were male. Using this information, it can be calculated that 55% of the female population was anti-MOG antibody positive at onset compared to 45% of the male population.

While relapses are not as common in ADEM as they are in MS, some patients will experience more demyelinating episodes following their initial ADEM presentation. Some of these patients (2) fit the MS criteria, but others who experienced relapses (4) did not and have been categorized under “relapsing non-MS.�

87% (54) of the total ADEM population, however, was diagnosed with Monophasic Acquired Demyelinating Syndrome (ADS). After their initial ADEM presentation, these patients did not experience new or worsening symptoms. Of these 54 patients, 26 were anti-MOG antibody positive while 28 were negative.

It is important to note while interpreting this data that this is the only chart that DOES include the low positive patients mentioned in the demographics slide.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

Of the 68 patients depicted in this pie chart, 43% (29) persisted negative across the duration of their follow-up appointments while 26% (18) persisted positive. 7% (5) of patients converted from positive to negative while another 7% (5) were initially low positive but then determined to be positive. 4% (3) of patients were positive but fluctuating. 2% (1) converted from negative to positive and another 2% (1) from low positive to negative. Finally, 9% (6) of patients did not have a follow-up appointment.

It is important to take away that over half of the patients who were positive at onset persisted positive throughout the duration of their follow-up period. In addition, 17% (3) of those who were persistently positive experienced a relapse at some point.

There are two different types of relapses one can experience: an MRI relapse, defined by the presence of new lesions in the brain and spinal cord, or a clinical relapse, defined by the expression of new symptoms. Of the 31 patients who were anti-MOG antibody positive at onset, 25.8% (8) experienced some type of relapse. Five had MRI relapse(s), two had clinical relapse(s), and one had both MRI and clinical relapse(s). Some of these relapses have been further characterized: the two clinical relapses were Optic Neuritis (ON) relapses, or damage to the optic nerves. The patient who experienced both MRI and clinical relapses had new lesions arise in the brainstem region and had ON. Of the 31 anti-MOG antibody negative patients, 12.90% (4) of patients experienced relapses: three patients with MRI relapse(s) and one with clinical relapse(s).

It is important to note that the number of patients who had clinical relapses expressed in the data may be lower than the actual number of patients who experienced clinical relapses, as some patients do not always mention new symptoms to their doctor, particularly as these patients are children.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

The graph above represents the concentration of the antibody in the persistently positive population vs. time in months since initial visit. Despite these patients being labelled “positive� for the antibody throughout the entirety of their follow-up period, there was an overall negative trend in the data as the concentration of the antibody decreased in nearly every patient. There was one outlier patient whose MOG antibody concentration increased over time.

Lesions, or scarred tissue, are commonly found in the brain and spinal cord of ADEM patients.

The chart above reveals the number of brain lesions each patient was determined to have. It is important to note while analyzing this chart that those counting the number of lesions capped off the counting at sixteen lesions, so it is likely that those who were said to have sixteen actually had more. Thus, the median lesion count is not an accurate representation of the actual number of lesions in the population.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

In the anti-MOG antibody negative group, 48.39% of patients had greater than fifteen lesions, compared to 70.97% of the anti-MOG antibody positive group. While the median number of lesions for both groups was greater than fifteen, the twenty-fifth percentile proves telling. In the positive cohort, the twenty-fifth percentile was greater than fifteen lesions, while in the negative cohort, it was two lesions. In addition, 29.03% of the negative population had between zero and four lesions, compared to 3.23% of the positive population.

Overall, patients in the anti-MOG antibody positive group had a greater number of lesions than the patients in the negative group. To test the statistical relevance of this data, a Mann-Whitney U test was conducted, and the p-value was .034; thus, it is safe to say that the difference in lesion count between the two groups is significant.

Eleven specific brain lesion locations were analyzed. They were the following: cerebellar, diencephalic, internal capsule, intracallosal, thalamic, basal ganglia, brainstem, periventricular, juxta cortical, peri 4th ventricular, and cerebellar peduncles. Across both the anti-MOG antibody positive and negative groups, the brainstem, juxta cortical, and periventricular lesions were the lesions most commonly observed. The juxta cortical lesion was the lesion most often observed in the anti-MOG antibody positive group (found in 80.65% of positive patients), and the brainstem lesion was the most common lesion in the negative group (found in 67.74% of negative patients).

The thalamic lesion had the greatest discrepancy between the positive and negative groups: 67.74% of positive patients had thalamic lesions compared to 38.71% of negative patients.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

Bilateral lesions, lesions expressed on both sides of the brain, are common in ADEM patients.

90.32% (28) of the anti-MOG antibody positive patients and 80.65% (25) of the anti-MOG antibody negative patients had bilateral lesion distribution.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

In addition to brain lesions, spinal cord lesions were observed in the ADEM patients. Nineteen patients in total had spinal cord lesions, 68% of whom were anti-MOG antibody positive. There are two main types of spinal cord lesions: longitudinal extensive transverse myelitis (LETM) lesions and focal lesions. By definition, LETM lesions extend over three or more consecutive vertebrae.

54% (7) of anti-MOG antibody positive patients with spinal cord lesions had LETM lesions, 31% (4) had focal lesions, and 15% (2) had both LETM and focal lesions. In the anti-MOG antibody negative cohort, 67% (4) had LETM lesions and 33% (2) had focal lesions.

There are different phenotypic expressions of ADEM episodes. Optic Neuritis (ON) and Transverse Myelitis (TM) are inflammation that damages the optic nerves and spinal cord respectively.

25% (9) of anti-MOG antibody positive patients expressed TM, 5% (2) expressed ON, and 3% (1) expressed both ON and TM. The remaining 67% (24) of the cohort had some “other” type of clinical episode that was not further categorized.

In the anti-MOG antibody negative group, 3% (1) of patients expressed ON and 3% (1) expressed TM. The other 94% (29) of patients fell under the “other” label.

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Analysis of Anti-MOG Data For Pediatric ADEM Patients | Simi Bleznak ’19

Patients with Lesion Enhancement

Lesion enhancing using Gadolinium can reveal a leakage in the blood brain barrier. It is not very common in ADEM. Four anti-MOG antibody positive patients had lesion enhancement compared to two anti-MOG antibody negative patients.

Summary

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ANOUSHKA GIDH ‘19 Anoushka Gidh, a senior from Harleysville, PA, has attended Baldwin since ninth grade. She is Co-Editor in Chief of The Baldwin Review and loves participating in the tennis and swim teams for Baldwin. She is Head of the Women on the Rise speaker series club and is a member of Baldwin’s Yearbook club PRISM, Lamplighters and Modern Science Club. She enjoys hiking and playing piano in her free time.

Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications

By Anoushka Gidh ’19 Wills Eye Hospital, Ocular Oncology Service, Thomas Jefferson University, Philadelphia, PA Summer 2018

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

INTRODUCTION: Anatomy of the Eye

Figure 1: Anatomy of Eye Diagram

Figure 2: Normal Optical Coherence Tomography Showing Fovea

The eye is composed of an anterior segment and a posterior segment (Figure 1). The portion near the front of the eye, called the anterior segment, extends from the ocular surface (corneal, scleral, and conjunctiva) to the lens and contains the aqueous humor, iris, and ciliary body.1 The portion near the back of the eye, called the posterior segment, extends from the posterior aspect of the lens to the back of the eye and contains the vitreous humor, retina, fovea, optic disk, and optic nerve.2

Layers of the retina can be visualized through optical coherence tomography (OCT).

This form of imaging is also important in assessing the fovea, a single, small depression in the retina that is important for visual acuity (Figure 2). From the top to the bottom, the posterior segment can be visualized starting from the vitreous humor (displayed as black space), the fovea (represented by the dip in the retina), and the choroid (the blood vessel-containing layer that lies below the bright line and is represented by a swiss cheese-like layer).3 Choroidal nevi and melanomas can be found in both the posterior and anterior segments.

CHOROIDAL NEVUS: Characteristics Choroidal nevi are benign lesions found in the choroid, a vascular layer found beneath the retina. Often described as a freckle in the eye, nevi are composed of melanocytes. Melanocytes, "Anterior Part of the Eye," Augenwissen INT, Accessed September 10, 2018, https://www.know-the-eye.com/ the-eye/anterior-part-of-the-eye/.

2 "Posterior Part of the Eye," Augenwissen INT, Accessed September 10, 2018, https://www.know-the-eye.com/ the-eye/posterior-part-of-the-eye/.

3 Hilary Wilson, "OCT Bootcamp: The Basics of Retinal OCT," Loma Linda Health (November 2008), Accessed September 10, 2018, http://lomalindahealth.org/media/health-care/pdfs/ophthalmology/oct-b.pdf.

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

pigmented cells responsible for skin color, can also be found in the choroid.4 In the eye, melanin typically creates a dark pigmentation, allowing choroidal nevi to be visualized.

In most cases, choroidal nevi are less than 2 millimeters in diameter, relatively the same size as the optic disk.5 In Figure 4, the lighter circle on the right side is the optic disk, and the darker circle closer to the left side is the nevus. The diameters of the optic disk and the nevus in this image are relatively the same size.6

Figure 3: OD Nasal Choroidal Nevus

Figure 4: OD Temporal Choroidal Nevus Complications

COMPLICATIONS

Nevi can be associated with the development of drusen, a collection of lipids and fatty proteins (Figure 5).

Choroidal nevi can also leak fluid and can cause the layer above the choroid to separate. As a result of fluid leakage, there is a risk of developing retinal detachment, which can contribute to vision loss.7 Finally, and most importantly, although benign, nevi have the potential to become cancerous and develop into a melanoma. Therefore, patients with choroidal nevi are strongly recommended to have annual eye examinations to monitor for the development of choroidal melanomas.8

Figure 5: Choroidal Nevus with Drusen

Jerry A. Shields and Carol L. Shields, Intraocular Tumors: An Atlas and Textbook (Philadelphia, PA: Wolters Kluwer, 2016), 69-80.

5 J. R. Lacey, "Choroidal Nevus: A Common Eye Condition That Can Become Lethal," Master Eye Associates (November 2013), Accessed August 21, 2018, http://www.mastereyeassociates.com/eye-care-news-blog/acommon-eye-condition-that-can-become-lethal.

6 Ocular Melanoma Foundation, "Understanding Choroidal Nevi," Ocular Melanoma Foundation, Accessed August 20, 2018, http://www.ocularmelanoma.org/choroidalnevus.htm 7 Lacey, "Choroidal Nevus: A Common Eye Condition That Can Become Lethal."

8 Ibid.

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

CHOROIDAL NEVUS DEVELOPING TO CHOROIDAL MELANOMA:

While the previously mentioned complications can occur with choroidal nevi, several symptoms for choroidal melanoma can occur when a choroidal nevus progresses into choroidal melanoma.

There are many Figure 6: Choroidal Nevus Developed to Choroidal Melanoma factors to consider when monitoring nevi. The transformation from nevi to melanoma can be considered when growth is noted. Nevi typically stay stable in size. Therefore, when growth is noted, it is likely that it has progressed into a melanoma. Tumor growth can be assessed through evaluating changes in tumor thickness and diameter. However, even before a nevus becomes melanoma, there are a few signs observed by ophthalmologists that can inform them of more careful observation.9 During their time working at the Wills Eye Hospital in Philadelphia, Pennsylvania, Drs. Carol and Jerry Shields developed a mnemonic, “To Find Small Ocular Melanoma-Using Helpful Hints Daily,” to describe pertinent features that can be found on nevi and are associated with risk of progression into melanoma.

Such features include: tumor thickness greater than 2 millimeters, presence of subretinal fluid, development of visual symptoms, presence of orange pigment, tumor margin 3 millimeters or less from the optic disc, hollow appearance on ultrasound, absence of halo (a depigmented ring that surrounds a pigmented nevi), and absence of drusen.10 Orange pigment is a sign of disruption in a retinal layer due to growth of an underlying mass. This layer, called the retinal pigment epithelium (RPE) layer, attaches the retina to the choroid. When a mass is growing in the choroid, it can get big enough to disrupt it, causing the immune cells, called macrophages, to clear the area and take up the cellular residue. This process, ultimately, leads to the orange pigment observed on the mass.11 These lesions can be detected with fundus autofluorescence, a special imaging technique used to evaluate ophthalmic conditions such as bright spots. It is important to monitor nevi for these features so that a risk of developing into a choroidal melanoma can be assessed. In addition to these features, other concerning findings include discoloration of the iris, irregularly shaped pupil, glaucoma, and hemorrhage. While these are not necessarily specific to choroidal melanoma, further examination is advised to find the cause, especially in patients with a documented history Ocular Melanoma Foundation, "Understanding Choroidal Nevi."

Jerry A. Shields and Carol L. Shields, Intraocular Tumors: An Atlas and Textbook 11 "Retinal Pigment Epithelium (RPE)," Foundation Fighting Blindness, Accessed September 3, 2018, https:// www.blindness.org/glossary/retinal-pigment-epithelium-rpe.

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

of a choroidal nevus.12 Figure 6 displays a case of a choroidal nevus transforming into choroidal melanoma. Note the orange pigmentation, lack of halo, and close proximity of less than 3 millimeters to the optic disc. The remaining features can be detected by other diagnostic tests, which will be discussed later.

CHOROIDAL MELANOMA: Characteristics

Choroidal melanoma is the most common primary malignant intraocular tumor and the second most primary malignant melanoma in the body after skin melanoma.13 This cancer has been found to be more prevalent in blue-eyed Caucasians under the age of forty years.14

Imaging

Figure 7: OCT Imaging Displaying Choroidal Melanoma Tumors with Subretinal Fluid

Figure 7 displays a set of OCT images of choroidal melanoma. The imaging modality can detect fluid under the retina— seen as a dark space within the deeper layer of the retina indicated by the red circles. In each of the pictures, a slight elevation can be noted in the layer of the retina.

This indicates an underlying mass consistent with a choroidal lesion, which, in this case, is a choroidal melanoma.

Paul T. Finger, "Choroidal Melanoma," New York Eye Cancer Center (June 16, 2016), Accessed August 19, 2018, https://eyecancer.com/eye-cancer/conditions/choroidal-tumors/choroidal-melanoma/.

13 Enrique Garcia-Valenzuela, "Choroidal Melanoma: Practice Essentials, Overview, Pathophysiology," Medscape (July 12, 2018), Accessed August 20, 2018, https://emedicine.medscape.com/article/1190564-overview.

14 Lacey, "Choroidal Nevus: A Common Eye Condition That Can Become Lethal."

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

Figure 8: Fundus Imaging of CM Tumor

Figure 9: Fluorescein Angiography of CM Tumor

Figure 8 displays a choroidal melanoma in a fundus image. Figure 9 shows the same mass under evaluation with fluorescein angiography. Fluorescein angiography is another imaging technique in which fluorescent dye is injected intravenously. The dye travels through the bloodstream to reach the blood vessels in the posterior segment of the eye. The special camera used in fluorescein angiography can capture the presence of the dye and displays it as bright areas. Taken seconds after the dye is injected, fluorescein angiography can provide a clear image of the choroidal mass and shows areas in which the dye has accumulated (Figure 9).15 These images show the comparison between a fundus image and a fluorescein angiography image.

Figure 10: Choroidal Melanoma Tumor Ultrasound

Figure 11: Choroidal Melanoma Tumor A-Scan

Figure 10 displays an ultrasound of a choroidal melanoma tumor. An ultrasound “uses sound waves to produce pictures of the inside of the body.”16 Choroidal melanoma are classically hollow tumors and do not create a shadow on ultrasound.

Amplitude scans, or A-scans, provide data on the length of the eye and are used “to determine the size and ultrasound characteristics of masses in the eye, in order to determine the Jerry A. Shields and Carol L. Shields, Intraocular Tumors: An Atlas and Textbook "Ultrasound (Sonography)." RadiologyInfo. March 9, 2018. Accessed September 9, 2018. https://www.radiologyinfo.org/en/info.cfm?pg=genus.

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

type of mass.�17 In the A-scan (Figure 11), the section of the tumor shows a hollow image, as the wavelengths decrease drastically after hitting the front of the tumor, and the wavelengths increase as the A-scan hits the retina. Other than choroidal melanomas, there are other masses that can grow in the choroid. Therefore, it is important to use different diagnostic techniques to help differentiate such masses. Ultrasound and A-scan imaging are effective tools in assisting doctors in differentiating the various diseases that can be found in the choroid.18 Choroidal hemangioma, for example, is a benign vascular tumor that is also found in the choroid. Choroidal hemangiomas can be detected by ultrasound (Figure 12) through the presence of a shadow, indicating that the tumor is solid. The A-scan (Figure 13) can further characterize the mass and presents as wavelengths that remain high, which also indicates a solid mass. These are some differentiating characteristics that will allow for proper diagnosis of an unknown choroidal tumor.

Figure 12: Choroidal Hemangioma Tumor Ultrasound

Figure 13: Choroidal Hemangioma Tumor A-Scan

GENETICS

Figure 14: Unbalanced Translocation

Figure 15: Monosomy Occurring During Meiosis II

"A-scan Ultrasound Biometry." Wikipedia. August 30, 2018. Accessed September 9, 2018. https://en.wikipedia.org/wiki/A-scan_ultrasound_biometry.

18 Ibid.

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

Every smoatic cell in the human body contains 23 pairs of chromosomes. Each chromosome has a p arm and a q arm and both arms are met at the centrosome. Pairs of chromosomes are homologous, which means they are similar. The homologous chromosomes have the same p and q arm lengths, and the centromere is at the same location. The DNA composition is also similar, one coming from each parent. Each of the 46 total chromosomes comes with a risk of mutating.19

Translocation occurs when homologous chromosomes exchange genetic information, but results in balanced or unbalanced chromosomes (Figure 14) and more genetic diversity.20

During meiosis, the pairs of homologous sister chromatids in each cell split twice to create four daughter cells, each of which should have one of the chromosome from each homologous pair. However, in some cases in either meiosis I or II, monosomy can occur. Monosomy is the condition of ending with four daughter cells, but one chromatid did not properly split, leaving one cell without a chromosome (Figure 15).21

Genetics of Choroidal Melanoma

The management and treatment of choroidal melanomas have been better understood through the emergence of genetic testing. With a blood test, doctors can determine if patients have a hereditary gene mutation that can increase the patient’s risk for developing choroidal melanomas as well as other cancers.22 Chromosome 3 contains one of those genes of interest, BAP-1. Associated with a tumor suppressor Figure 16: Monosomy 3 gene, mutations in BAP-1 has been correlated with malignant mesothelioma, renal cell carcinoma, and choroidal melanoma. The risk of developing these cancers are particularly high when one of the two chromosomes are lost— leading to one chromosome (a monosomy). In addition to an increased risk of developing other cancers, patients with monosomy 3 have a strong association with metastasis, which can lead to death. This mutation is present in approximately 30-50% of all uveal melanomas (Figure 16).23

In addition to chromosome 3, chromosomes 6 and 8 can also determine the prognosis of patients affected by uveal melanoma. During translocation, a chromosome can either gain or lose a part of its arms. For example, chromosome 6 gain is almost never associated with monosomy 3, which is an indicator of good prognosis of the uveal melanoma, because it is mutually exclusive with monosomy 3. The gain of genetic information on the q arm of chromosome 8 has also been in studies as an indicator of poorer prognosis due to its common association with monosomy 3.24 "What Is a Chromosome?" U.S. National Library of Medicine, Accessed September 18, 2018, https://ghr.nlm. nih.gov/primer/basics/chromosome.

20 "Chromosomal Translocation," Wikipedia (September 11, 2018), Accessed September 18, 2018, https:// en.wikipedia.org/wiki/Chromosomal_translocation.

21 "Meiosis," Khan Academy, Accessed September 18, 2018, https://www.khanacademy.org/science/biology/ cellular-molecular-biology/meiosis/a/phases-of-meiosis.

22 Erin E. Nichols, Ann Richmond, and Anthony B. Daniels, "Tumor Characteristics, Genetics, Management, and the Risk of Metastasis in Uveal Melanoma," NCBI (April 29, 2016), Accessed July 30, 2018, https://www. ncbi.nlm.nih.gov/pmc/articles/PMC5526754/.

23 Erin E. Nichols, Ann Richmond, and Anthony B. Daniels, "Tumor Characteristics, Genetics, Management, and the Risk of Metastasis in Uveal Melanoma."

24 Ibid.

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Complications

If not managed properly, choroidal melanoma can metastasize to other parts of the body.25 If found in the posterior segment (especially near the fovea), the tumor could obstruct the retina and cause flashes, floaters (black dots seen in the vision), or even a loss of vision due to partial or full retinal detachment. Retinal detachment is possible if fluid leaks to behind the retina. If found in the anterior segment, the tumor can push on the natural lens and cause irregular astigmatism, an irregular curvature of the lens which causes blurry vision.

Neovascularization, an abnormal growth of blood vessels, can be observed using fluorescein angiography. Visual complications include blurry vision and seeing floaters. Other complications include retinal pigments, orange discoloration, exudative retinal detachment, subretinal hemorrhaging, or vitreous hemorrhaging.26

Furthermore, the risk of metastasis, although relatively small, is possible. The tumors can metastasize through the lymph nodes to distant sites in the body or to the body’s organs.27 Usually, the liver, lungs, bones, and brain are most often involved with metastases. Unlike skin melanomas, choroidal melanomas are rare. In fact, there are approximately 2,500 cases of choroidal melanoma in the United States annually, most of which are treatable.

Treatments

Plaque radiotherapy (Figure 17) works to shrink the choroidal melanoma and kill the cancerous cells through radiation. The treatment involves a gold piece of metal embedded with radioactive seeds that is surgically placed over the eye. Radiation from the seeds will be aimed at the tumor to produce its therapeutic effect.28 Thus, prior to the procedure, it is important to locate the tumor and determine its exact dimensions. During the day of the procedure, the patient is taken into the operating room and given local anesthesia to the eye prior to incision. In order to place the plate over the tumor, an incision is made over the outer layer of the eye, the conjunctiva.

Figure 17: Plaque Radiotherapy Treatment This incision creates a pocket to allow the plate to be inserted over the tumor. The metal plate is sewn onto the eye to allow the radioactive seeds to kill the cancerous cells.29 After a few days of radiation exposure, the plaque is surgically removed. Following therapy, a follow-up with the doctor is important to monitor treatment response of the tumor.

Transpupillary thermotherapy (TTT) works in a similar way to plaque radiotherapy, where the objective is to kill the cancerous cells. With TTT, the patient should undergo a similar set-up, ending in the operating room. This method of treatment involves exposing the tumor to a laser "Risk Factors for Growth and Metastasis of Small Choroidal Melanocytic Lesions," Egyptian Journal of Medical Human Genetics (October 3, 2013), Accessed August 4, 2018, https://www.sciencedirect.com/science/ article/pii/S0161642095308640.

26 Enrique Garcia-Valenzuela, "Choroidal Melanoma: Practice Essentials, Overview, Pathophysiology."

27 "Risk Factors for Growth and Metastasis of Small Choroidal Melanocytic Lesions."

28 "NCI Dictionary of Cancer Terms," National Cancer Institute, Accessed August 19, 2018, https://www.cancer. gov/publications/dictionaries/cancer-terms/def/plaque-radiotherapy.

29 "NCI Dictionary of Cancer Terms."

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Choroidal Nevus and Choroidal Melanoma: Characteristics, Treatments and Complications | Anoushka Gidh ’19

at a low level setting through the opening of the eye, the pupil. This works to kill the cancer cells and shrink the tumor. For a more effective and thorough approach, doctors may do a combined treatment, using both plaque radiotherapy and TTT (Figure 18).30

If the tumor has progressed and spread drastically, the eye may have to be enucleated Figure 18: Combined Plaque Radiotherapy and TTT to prevent the risk of it metastasizing to the rest of the body. Enucleations of the eye is a procedure done if the melanoma cannot be treated by any other way. Usually melanomas do not spread into the optic nerve, but it could severely damage the nerve, causing very poor visual acuity. A few months after an enucleation, the patient can opt to get a prosthesis.

Treatment Complications

With any treatment comes possible complications. With combined plaque radiotherapy and TTT, the level of intensity of killing cancerous cells could also kill normal and healthy cells. This could lead to poor visual acuity which could even mean 20/200 or worse. This happens to 64% of the patients five years after the treatment. Many other complications include “retinopathy, maculopathy, papillopathy, macular and extramacular retinal vascular obstruction, surface wrinkling retinopathy, rhegmatogenous retinal detachment, vitreous hemorrhage, cataract, and neovascular glaucoma, retinal exudative, retinal edema, nerve fiber layer infarction, or vascular sheathing.”31

Furthermore, enucleation can come with the complication of becoming infected. This could lead to severe health issues for the patient. Additionally, discharge can occur when the patient wears a prosthesis.

CONCLUSIONS:

While choroidal nevus and choroidal melanoma can both be rare and severe diseases, with the right doctor care, the diseases can be treated. Annual eye check-ups and examinations are vital for the health and well-being for anyone.

ACKNOWLEDGMENTS:

Thank you to Carol Shields, MD, Jerry Shields, MD, Michael Chang, MD, Sara Lally, MD, Arman Mashayekhi, MD, Sandra Dailey, Wills Eye Hospital’s Oncology Service Faculty and Staff, and my family for supporting me throughout my research and allowing me to spend six weeks shadowing in the Wills Eye Hospital clinics and operating rooms this past summer.

Kathleen Koviak, "Transpupillary Thermotherapy," Kellogg Eye Center (May 2015), Accessed July 30, 2018, http://www.med.umich.edu/1libr/Ophthalmology/OcularOncology/TranspupillaryThermotherapy.pdf.

31 Carol L. Shields, "Combined Plaque Radiotherapy and Transpupillary Thermotherapy for Choroidal Melanoma," JAMA Internal Medicine (July 1, 2002), Accessed August 4, 2018, https://jamanetwork.com/ journals/jamaophthalmology/fullarticle/271249.

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ANISHA DEVAS ’19 Anisha Devas is a senior from Exton, PA, and has attended Baldwin since ninth grade. Outside of the classroom, she is a senior head of the Teens Aware of Cancer club and the Lamplighters Communication Team. She is also actively involved as a Writing Center tutor, a member of the B-Flats a cappella group and a student ambassador of the Alex’s Lemonade Stand Foundation. In her free time, she enjoys taking pictures, volunteering at a local hospital and spending time with friends and family.

QUANTIFICATION OF ORGANIC COMPOUNDS’ HYDROGEN BONDING STRENGTH AS HYDROGEN BOND DONORS

By Anisha Devas ’19

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Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors | Anisha Devas ’19

ABSTRACT:

Using a UV-vis spectrophotometer, twenty-nine organic compounds were tested for their ability and strength of hydrogen bonding, specifically acting as donors. These compounds LUMOlowering abilities (explained more later) and tight desolvation in water as a result of hydrogen bonding allow them to serve as catalysts and improve drug design.1 This is because more hydrogen bonding causes more separation of charges and allows for higher frequency of energy to be absorbed, which allows reactions to happen faster, such as drug delivery. The measured and calculated data establishes wavelength shift as a reliable way to predict hydrogen bonding abilities across various kinds of compounds.

INTRODUCTION:

Hydrogen bonding is an intermolecular force that results from the attraction between the partially positive hydrogen atom of one molecule and the partially negative nitrogen, oxygen, or fluorine atoms of another molecule. Hydrogen bonding is a major component of our world’s existence through its crucial roles in water’s high specific heat capacity and strong surface tension, DNA replication, and drug design. More specifically related to drug design, stronger hydrogen bond donors can dissolve in water more. This causes higher desolvation energies, which are responsible for a compound’s ability to permeate through cell membranes.2 Therefore it is imperative to be able to uniformly measure and compare various compounds’ ability to be hydrogen bond donors to design the most effective medicine.

Originally, pKa, the acid dissociation constant at a logarithmic scale, was believed to be sufficient for predicting and comparing organic compounds’ hydrogen bonding abilities. This is because acidity measures the concentration of the hydrogen ions and when acids dissociate, they release their hydrogen ion(s), showing their ability to be hydrogen bond donors. However, while pKa values can technically be used to deduce hydrogen bonding abilities, they neglect secondary interactions and binding geometry, only making them useful in comparing compounds that have similar structures.3 In contrast, this UV-vis method for measuring hydrogen bonding abilities can analyze a wide range of organic compounds, including the sensitive weak hydrogen bonding compounds.

Pierrik Lassalas, Bryant Gay, Caroline Lasfargeas, Michael J. James, Van Tran, Krishna G.Vijayendran, Kurt R. Brunden, Marisa C. Kozlowski, Craig J. Thomas, Amos B. Smith, III, Donna M. Huryn, and Carlo Ballatore “Structure Property Relationships of Carboxylic Acid Isosteres.” Journal of Medicinal Chemistry, March 11, 2016, [3189 ]DOI: 10.1021/acs.jmedchem.5b01963 2 Ibid.

3 Ryan R. Walvoord, Phuong N. Huynh, and Marisa C. Kozlowski, "Quantification of Electrophilic Activation

by Hydrogen-Bonding Organocatalysts," Journal of the American Chemical Society, October 17, 2014, [16055], DOI:10.1021/ja5086244.

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Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors | Anisha Devas ’19

At the core of this method’s ability to test a wide range of organic compounds lies the sensor. The sensor, imidazopyrazinone S, was chosen because it can serve as a hydrogen bond acceptor due to its oxygen atom with lone pairs. Additionally, it displays the property of solvatochromism, which is color change that results from a change in polarity.4 Along with this key quality, the LUMO-lowering abilities of compounds made it possible to use absorption and wavelength values measured by the UV-vis spectrophotometer. The hydrogen bond interaction stabilizes the highest occupied molecular orbital (HOMO) of the sensor to a greater degree than the lowest unoccupied molecular orbital (LUMO).5 The change in the gap between the two levels results in change of the type of energy absorbed. Specifically, increasing the HOMO-LUMO gap results in a hypsochromic shift (blue shift), meaning the maximum absorption value shifts toward a shorter wavelength and higher frequency of energy.6 As a result, there is an observable color change from pink to yellow in the sensor solution as the hydrogen bond donor is titrated.

Figure 2: The sensor is dissolved in DCM and accepts the hydrogen bond from the titrated compound (the hydrogen donor, H-D).

METHOD:

The method uses a pyrazinone sensor that serves as a hydrogen bond acceptor due to its oxygen atom with lone pairs and a hydrogen bond donor (as shown in Figure 1), which is titrated into the sensor. After each titration, the UV-vis spectrophotometer measures the change in the wavelength at the highest absorption value. However, to achieve this, a series of steps must be taken first: 1. Analyze the structure and atomic mass of the hydrogen bond donor that will be tested in the trial, comparing it to other molecules reactivity and masses to determine how much of the molecule will dissolve in the sensor solution. The molecule, whether a solid or liquid, will be placed in a five milliliter volumetric flask with enough distilled dichloromethane (DCM) to "Solvatochromism." Wikipedia. Accessed August 14, 2018. https://en.wikipedia.org/wiki/Solvatochromism.

Ryan R. Walvoord, Phuong N. Huynh, and Marisa C. Kozlowski, "Quantification of Electrophilic Activation

by Hydrogen-Bonding Organocatalysts," Journal of the American Chemical Society, October 17, 2014, [16057], DOI:10.1021/ja5086244.

6 Ryan R. Walvoord, Phuong N. Huynh, and Marisa C. Kozlowski, "Quantification of Electrophilic Activation

by Hydrogen-Bonding Organocatalysts," Journal of the American Chemical Society, October 17, 2014, [16056], DOI:10.1021/ja5086244.

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Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors | Anisha Devas ’19

2.

3.

4.

5.

6.

7.

make a 5 milliliter solution. This information is also used to come up with an equivalence ratio between the hydrogen bond donor and sensor solution to make sure enough donor is being added to see changes in the wavelength of the sensor.

Using a ten milliliter volumetric flask, dissolve four milligrams of the sensor solution in enough distilled DCM to make a ten milliliter solution. Then, in a 2 milliliter volumetric flask, dissolve twenty-five microliters of the solution in more distilled DCM to make two milliliters of a diluted solution, the sensor. Mark the bottom of the meniscus of the sensor using a sharpie marker.

Next, procure two cuvettes. Using a full one milliliter syringe, place the diluted solution into the first cuvette, and fill the second cuvette, two-thirds of the way, with distilled DCM to make sure that the light passes through the solution and not air. The second cuvette acts as the blank and calibrator, eliminating biases that could be caused by any remaining impurities in the DCM. Run the blank cuvette in the UV-vis spectrophotometer for a baseline. Then run the cuvette with the sensor solution to determine at what absorbance value the maximum wavelength of the solution occurs, which should respectively be approximately 0.3 and 490 nanometers.

Using the micropipette, add the appropriate equivalence of hydrogen bond donor. Then, using argon gas, some of the volatile DCM is evaporated down to the sharpie line to keep the concentration constant, and therefore by Beer’s Law, the absorbance will also remain constant, preventing any biased change in the wavelength.

Place the cuvette with the sensor and hydrogen bond donor into the spectrophotometer and check if the change in wavelength is over ten nanometers. If so, the trial will need to be restarted because such a large change results in an imprecise titration graph.

Continue to add hydrogen bond donor solution at the same equivalence to the cuvette with the sensor solution until the change in wavelength between two runs of the spectrophotometer determines a change less than 0.2 nanometers.

At this point, double the equivalence and keep adding the hydrogen bond donor solution until the wavelengths change is again less than 0.2 nanometers, meaning the sensor is fully saturated with hydrogen bonds, which can also be seen from the sensor’s color change.7

Ryan R. Walvoord, Phuong N. Huynh, and Marisa C. Kozlowski, "Quantification of Electrophilic Activation

by Hydrogen-Bonding Organocatalysts," Journal of the American Chemical Society, October 17, 2014, [16056], DOI:10.1021/ja5086244.

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Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors | Anisha Devas ’19

RESULTS:

Once the wavelength values no longer change, the absorbance and wavelength values can be used to make a titration graph to assess the accuracy of the data and predict other values. These measured values are also used to make calculations for the wavelength shift, the equivalence equilibrium value, the equilibrium constant value, K, and the natural log of K. The natural log of K is particularly useful because it relates directly to ΔG and has a correlation with the wavelength shift as shown in Figure 6. This means the total wavelength shift value can be used to determine which compounds have the most separation and most binding energy and are therefore the best hydrogen bond donors.

Since many compounds were tested using this method, it would be impossible to mention the results of every trial. However, thiophenols are an important group of molecules that were tested this past summer.

These compounds are made of the thiofunctional group (SH), which is attached to a benzene ring. When the UV-vis method was applied on these thiophenols, no significant amount of wavelength shift was observed even after the addition of thousands of equivalence of the thiophenol. After testing several thiophenols, it was concluded that since the sulfur-hydrogen bond in thiophenols were not as polarized as an oxygen-hydrogen bond. This means the sulfur holds a better negative charge than oxygen, and the sulfur is therefore more stable and does not want to separate its charges, which is necessary for hydrogen bonding. For this reason, most thiophenols were not efficient hydrogen bond donors.

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Quantification of Organic Compounds’ Hydrogen Bonding Strength as Hydrogen Bond Donors | Anisha Devas ’19

FUTURE DIRECTIONS:

The next step of this study is to test more compounds and create a chart of good hydrogen bond donors, which will be especially useful for improving drug design. Additionally, some of the methodology could be improved. With the current method, only uncolored compounds could be fully and accurately tested because occasionally the absorbance values would continually rise too high when a colored compound was tested. It was also difficult to dissolve some compounds in the DCM, so it might be necessary to find an alternate solvent to test more variety of compounds.

Minor improvements will allow for more compounds to be tested with greater certainty, improving the validity of the study and future drug design.

ACKNOWLEDGEMENTS:

I would like to thank the Chemistry Department at The University of Pennsylvania and specifically Dr. Marisa C. Kozlowski, Thomas Paniak, and the entire Kozlowski group for teaching and guiding me this summer. Thank you to Mrs. Davis for connecting me with this research opportunity and allowing me to explore more fields of Chemistry.

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ALEXA DIECIDUE ’19 Alexa Diecidue of Villanova, PA, is a senior at Baldwin and has been at the School since Pre-K. She is heavily involved in student leadership, currently holding the position of Upper School Senate Head. Alexa is also a co-head of Florilège, Baldwin’s French literary magazine. She enjoys participating in several clubs such as Mock Trial, Cradles to Crayons, Girls Learn International, Lamplighters and Peer Counseling. Alexa is a captain of both the tennis and swim teams.

Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer Alexa Diecidue ’19, Dr. Ayesha Shafi PhD Sidney Kimmel Cancer Center, Karen Knudsen Laboratory Department of Cancer Biology

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Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer | Alexa Diecidue ’19

INTRODUCTION:

Prostate Cancer (PCa) is a slow-growing disease that mostly affects men over the age of 50. It is the most common cancer in American men, making it an important disease to study in order to develop more effective treatment. PCa is an androgen-dependent disease, meaning it requires androgen hormones to grow and metastasize.1 Androgen Deprivation Therapy (ADT), is commonly used to treat metastatic PCa. This reduces the levels of androgen hormones with drugs or surgery to prevent cancer cell growth. Although initially effective, it is common that tumors recur 2-3 years post ADT and the cancer is then deemed castration-resistant prostate cancer (CRPC). As of now, there is no effective cure for CRPC, which emphasizes the need for new treatments in advanced PCa.

Several studies demonstrate that disturbances in circadian rhythms, like shift work, jet lag, lack of sleep, and exposure to light at night are all associated with increased risk of PCa.2 The circadian clock is an inherited molecular timekeeping mechanism that regulates many daily biological processes and behaviors. Its machinery is an auto-regulatory network made up of transcription-translation positive and negative feedback loops. The positive pieces of the feedback loop control expression of the key genes that make up the negative components of the loop: cryptochrome (CRY1 and CRY2) and period (PER1, PER2, and PER3) genes. The CRY-PER genes enter the nucleus to repress positive activity, thus creating daily rhythmicity of metabolic, cellular, and physiological functions for homeostasis. Importantly, circadian factors are involved in functions such as the DNA damage response (DDR) network.3 This includes DNA repair, DNA damage checkpoints, and apoptosis. Specifically, CRY1 has been shown to be elevated in metastatic PCa. However, the role that CRY1 plays in PCa and DDR is unknown. Thus, this study focuses on elucidating the role of CRY1 in DNA damage response in PCa and hypothesizes that CRY1 is a crucial component of DDR.

MATERIALS AND METHODS: Western Blot Analysis

22Rv1 cells were lysed by four rounds of freeze/thaw treatment using RIPA lysis buffer.

Protein concentration was quantified using the Lowry assay. A 10% SDS gel was used to resolve 40 μg of protein. Proteins were then detected using CRY1 and Vinculin antibodies at a 1:1000 dilution overnight. Proteins were detected using ECL reagents (GE Healthcare) and exposed on film. ImageJ densitometry software was used to quantify protein expression.

RNA Isolation, Quantitative PCR, and cDNA:

RNA was isolated from tissue using TRIzol (Life Technologies #15596018). cDNA was generated using SuperScript VILO (ThermoFisher). Quantitative PCR was conducted with primers A. Shafi, A. E. Yen, and N. L. Weigel, "Androgen receptors in hormone-dependent and castration-resistant prostate cancer," Pharmacology and Therapeutics 140, no. 3 (2013).

2 M. G. Wendeu-Foyet and F. Menegaux, "Circadian Disruption and Prostate Cancer Risk: An Updated Review of Epidemiological Evidences," Cancer Epidemiology, Biomarkers & Prevention 26 (2017).

3 M. J. Schiewer and K. E. Knudsen, "Linking DNA Damage and Hormone Signaling Pathways in Cancer," Trends in Endocrinology and Metabolism 27, no. 4 (2016).

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Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer | Alexa Diecidue ’19

for common DDR genes (p21, FAS, Gadd45a, and 18S) with PowerSybr and an ABI StepOne machine according to manufacturer’s specifications.

Immunofluorescence (IF)

Cells were seeded onto coverslips and treated with radiation. Cells were harvested at varying time points and fixed in 3.7% formaldehyde. Next, cells were permeabilized in 0.5% TritonX-100 at room temperature for 15 minutes then blocked with 2% goat serum for 30 minutes at room temperature followed by staining with primary antibody (1:500) for 2 hours at 37°C. Then, the cells were washed with 1XPBS 3 times and stained with secondary antibody (1:1000) for 1 hour at 37°C. All IF coverslips were counterstained with DAPI to visualize nuclei, and then mounted on slides using Gelvatol. Foci were imaged on a Zeiss Confocal Laser Scanning microscope and analyzed using Fiji software for foci per cell.

RESULTS: DNA damage increases expression of CRY1 in PCa cells

To characterize the effect of DNA damage on CRY1 expression in PCa, 22Rv1 cells were treated with radiation (IR). First, cells were treated with increasing doses of IR, (i.e. 2Gy, 5Gy, and 10Gy), to induce DNA damage. The expression of the CRY1 protein increased directly with dosage as compared to the vehicle treated cells (Veh) (Figure 1A). Next, the effect of DNA damage over time was assessed. In Figure 1B, 22Rv1 cells were treated with a dose of 5Gy IR for 2 hours, 4 hours, and 8 hours. CRY1 protein expression was detected via Western blot. Over time, IR increased CRY1 expression as compared to Veh. In sum, DNA damage increases CRY1 expression in PCa cells.

A

B

FIGURE 1: DNA damage increases expression of CRY1 in PCa cells. A. 22Rv1 cells were treated with increasing doses of IR (2Gy, 5Gy, and 10Gy). Cells were harvested for protein. CRY1 and Vinculin protein expression was detected via Western blot. B. 22Rv1 cells were treated with 5Gy IR for 2, 4, and 8 hours. Cells were harvested for protein. CRY1 and Vinculin protein expression was detected via Western blot.

CRY1 is necessary to regulate DDR in PCa cells

To examine the role of CRY1 in DDR, CRY1 expression was knocked down in 22Rv1 cells.

Briefly, in this cell model, shCon represents the control cells with normal levels of CRY1 expression and shCRY1 represents cells with knocked down expression of CRY1 in 22Rv1 cells. Using this model, cells were treated with 5 Gy IR for 24 hours in both shCon and shCRY1 conditions. Cells were harvested for RNA to detect the effect of CRY1 knockdown on DDR genes, (p21, FAS, and

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Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer | Alexa Diecidue ’19

Gadd45a). In the positive control, radiation significantly increased expression of p21, FAS, and Gadd45a. Interestingly, when CRY1 is knocked down and treated with IR, the induction of the DDR genes are significantly reduced. This indicates that CRY1 is important for canonical DDR and has the potential to be an upstream regulator in PCa.

FIGURE 2: CRY1 is necessary to regulate DDR in PCa. CRY1 expression was knocked down in 22Rv1-shCRY1 cells for 72 hours and then cells were treated with 5 Gy IR for 24 hours. Cells were harvested for RNA. p21, FAS, Gadd45a, and 18S mRNA was detected. * p<0.05.

KNOCKDOWN OF CRY1 IMPACTS THE HOMOLOGOUS RECOMBINATION MEDIATED DDR PATHWAY IN PCA CELLS:

Since CRY1 is necessary for normal DDR, it is important to determine the specific repair pathway CRY1 influences. Radiation treatment causes double-strand breaks (DSBs), which induce a response in either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways of DDR7. DNA damage causes an increase in γ-H2AX foci formation, which is resolved within 24 hours in normal functioning cells. Additionally, Rad51 foci formation specifically represents DNA damage through the HR pathway. To elucidate the role of CRY1 in HR, CRY1 expression was knocked in PCa cells and then treated with 5 Gy IR for 2, 4, and 24 hours. Rad51 and γ-H2AX foci formation was detected using immunofluorescence (IF) along with DAPI staining to visualize the nuclei of cells. After IR treatment, there is a peak of DNA damage as detected through γ-H2AX foci formation at 2-4 hours in both the control and CRY1 knockdown cells, which is resolved by 24 hours (Figure 3). Interesting, while Rad51 foci formation peaks at 2 hours and is resolved by 24 hours in control cells, the CRY1 knockdown cells have a delay in Rad51 foci formation and continue to increase by 24 hours. This indicates that when cells lack CRY1, the cells are unable to repair damage through the HR pathway and the DNA damage persists. These data suggest that CRY1 is critical for HR mediated DDR in PCa cells.

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Cryptochrome 1 (CRY1) Modulates DNA Damage Response (DDR) in Prostate Cancer | Alexa Diecidue â&#x20AC;&#x2122;19

FIGURE 3: CRY1 regulates HR mediated DDR pathway in PCa cells. CRY1 expression was knocked down in 22Rv1shCRY1 cells for 72 hours and then cells were treated with 5 Gy IR for 2, 4, and 24 hours. Cells were fixed at the indicated time points, stained with Îł-H2AX and Rad51 antibodies, and imaged by confocal microscopy. Foci were counted and plotted as foci per cell for each time point.

DISCUSSION:

This study focused on elucidating the role of CRY1 in DDR in PCa using several in vitro models. Western blot, RNA isolation, qPCR analyses, and immunofluorescence techniques were utilized to address the hypothesis. The data indicated that DNA damage through IR increased CRY1 expression over time in PCa cells. Additionally, models derived to regulate CRY1 expression demonstrate that CRY1 knockdown compromises DNA repair. Specifically, CRY1 regulates HR mediated DDR in PCa cells. Future studies will investigate the role of CRY1 in other pathways for DDR including NHEJ.

These findings suggest a potential novel role for CRY1 as a key modulator of DDR impacting PCa survival, and that delineating the underlying molecular mechanisms of CRY1 and DDR interplay will allow for improved management of PCa.

ACKNOWLEDGEMENTS:

I want to thank Dr. Ayesha Shafi for mentoring me at Jefferson, teaching me about her work in Prostate Cancer, and showing me numerous experiments and techniques throughout the summer. Thank you to Dr. Knudsen for allowing me to spend time in her lab and introducing me to Dr. Shafi. I extend my thanks to everyone in the lab for being so friendly and welcoming, and for making me feel like part of the team. Lastly, I want to thank the Baldwin Science Department for supporting my research and fostering my interest in the sciences.

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MERIEL DOYLE ‘19 Meriel Doyle is a senior at Baldwin from Villanova, PA. She has attended Baldwin since Pre-K. Outside the classroom, she is a head of Baldwin’s French magazine Florilège, a member of the Lamplighter’s Communications Team and a head of Modern Science Club. She enjoys participating in Baldwin’s tennis, swimming and lacrosse teams, and in her free time, she plays the violin.

Construction of a Chimeric Adeno-associated Virus 2/Human Parvovirus 4 Vector for Gene Therapy Meriel Doyle ’19 University of Pennsylvania Gene Therapy Lab

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Construction of a Chimeric Adeno-associated Virus 2/Human Parvovirus 4 Vector for Gene Therapy | Meriel Doyle ’19

INTRODUCTION:

Gene therapy utilizes the infectious nature of human viruses and their ability to act as vectors for delivery of genetic material into targeted human cells. In the best circumstance, a viral vector should be able to deliver the recombinant DNA to the expected tissue specifically while also inducing transgene expression without eliciting a substantial immune response. Due to the diversity of the targets of gene therapy, there is no one vector that would be suitable for all situations. In the search for better, more efficient vectors, researchers are trying to incorporate the most useful features of different viruses to create hybrid vectors.

Although adeno-associated virus (AAV) vectors have recently proved to be a safe alternative to the widely used retroviral and adenoviral vectors, the vector could be improved in multiple aspects. In response to vector transduction, the human body produces neutralizing antibodies that block the AAV from entering into human tissues. In addition, the AAV genome size is small and the specificity of its tropism may be further improved. Upon infection, the virus tends to disperse broadly, and as a result, the specific gene expression is limited.

The construction of recombinant viral vectors has the potential to produce vectors with lower immunogenicity as well as highly specific tissue tropisms. The ability to create a chimeric adeno-associated virus/human parvovirus 4 vector would be advantageous in the possible applications for gene therapy. In previous studies, parvovirus B19 and human bocavirus 1 have already been shown to successfully package the AAV genome1,2. Since PARV4 was classified between B19 and human bocavirus 1 phylogenetically, it might be similarly useful. The expected hybrid vector will contain a PARV4 capsid that packages the AAV genome. By combining the advantages of AAV and PARV4, a novel viral vector will be created that might exhibit higher transduction efficiency and lower immune response. The PARV4 genome size is ~20% larger than AAV alone, which will allow the viral capsid to store a larger genome size. The specific tissue tropisms and the degree of immune response of PARV4 vectors are not yet known, so after successful construction of the chimeric vector, these aspects will be further evaluated.

MATERIALS AND METHODS:

To create the chimeric vector, a series of protocols was followed. In some vector production, a polymerase chain reaction (PCR) amplifies a gene of interest, but in this case, the PARV4 VP1 DNA fragment was synthesized by Thermo Fisher Scientific, so no PCR reaction was necessary.

Backbone and DNA fragment digestion. Restriction enzymes were used to digest the 3μg DNA plasmid (as well as the intended plasmid backbone) by the following reaction: 2 μL BamHI and 2 μL of Sal I with 36μL H 2O , 5μL DNA and 5 μL 10X Cutsmart Buffer (total 50 μL) were used to digest both the pshuttle.CMV.WPRE.SV40pA backbone and the PARV4 VP1 insert to create compatible “sticky ends” that could be ligated together to make a circular plasmid. The digestion was incubated at 37°C for 2 hr. The product was analyzed with 1% agarose gel electrophoresis and Ponnazhagan Selvarangan, Kristen A. Weigel, Sudhanshu P. Raikwar, Pinku Mukherjee, Mervin C. Yoder, and Arun Srivastava. "Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes." Journal of Virology 72, no. 6 (June 1998): 5224-30.

2 Ziying Yan, Nicholas W. Keiser, Yi Song, Xuefeng Deng, Fang Cheng, Jianming Qiu, and John F. Engelhardt. "A Novel Chimeric Adenoassociated Virus 2/ Human Bocavirus 1 Parvovirus Vector Efficiently Transduces Human Airway Epithelia." The American Society of Gene and Cell Therapy 21, no. 12 (December 2013): 2181-94.

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the bands with the correct sizes were purified. Following gel purification protocol, the purified product was eluted in 50 μL H2O .

Ligation. The ligation reaction was as follows: 1μL backbone DNA, 4μL insert, 2μL 10X ligation buffer, 1μL T4 Ligase, and 12 μL H2O . at 37°C for 1 hr.

Transformation. 2μL ligation product was used to transform the Stbl2 competent cells.

During transformation, the recombinant plasmid DNA was inserted into the Stbl2 competent cells.

After 30 min incubation on ice, the plasmid DNA and the bacteria were given heat shock in a 42°C water bath for 25 seconds so the bacteria would be able to take up the plasmid. The competent cells were put on ice for 2 mins for initial recovery, and then 0.9 mL SOC media was added to fully recover the cells. The tubes were placed in the shaker at 37°C for 1hr 30 min at 250 rpm.

The backbone of the plasmid contained a Carbenicillin resistance cassette; therefore, the bacteria were grown on a on a plate containing Carbenicillin in order to see which bacteria successfully took up the plasmid (the bacteria without a plasmid will not grow). 200 μL of the culture was grown in a plate containing 20 mL luria broth (LB) with 1.6% agar and 10 mg/L Carbenicillin. Six colonies were picked from the plate for minipreps after overnight incubation. It is important to pick multiple colonies due to the possibility that a plasmid may ligate the insert backwards, or the plasmid may not contain an insert at all.

Mini DNA preparation. After the bacteria culture was harvested, the pshuttle.CMV.PARV4P1.WPRE.SV40pA DNA plasmids from the bacteria were extracted using a Qiagen mini-prep kit. 1.2-1.5 mL bacteria culture was centrifuged at 14K for 1 min. After the supernatant was removed, 250 μL P1 Buffer was added and the pellet was resuspended in the buffer by vortexing. 250 μL P2 lysis Buffer was added and the tubes were inverted 5-6 times. After incubating for 5 min, 350 μL N3 neutralization Buffer was added and the tubes were inverted 5-6 times. The tubes were centrifuged at 14K for 3 min. After 3 min, the supernatant passed through columns attached to a vacuum. 750 μL PE Buffer was added to wash the DNA when passed through the column. The column was placed in an elution tube and centrifuged at 14K for 1 min.

After discarding the mini filter tube and replacing it with a new tube, 50 μL H2O was added to the column and centrifuged at 14K for 1 min. The purified pshuttle.CMV.PARV4VP1.WPRE.SV40pA DNA plasmids were retained in the tube after centrifugation.

Confirmation of mini DNA extraction. To check that the plasmids from the bacteria contained the intended insert, a small sample of the plasmids were digested with 0.5 μL BamHI, 0.5 μL Sal I, 12.5 μL H 20 , and 1.5 μL 10X Cutsmart Buffer (total 15 μL) to separate the PARV4 VP1 insert and the pshuttle.CMV.WPRE.SV40pA backbone. A 1% gel was run to check for two individual bands, which indicated that there were two strands of DNA present in each of the samples, and the DNA was extracted using gel purification protocol. Since the plasmids did contain the correct insert, the DNA was sent for Sanger sequencing to see that the nucleotides were in the correct order.

Transfection. HEK293 cells are the most commonly used cells for transfection in laboratories because they are easy to grow in a cell culture medium. To complete transfection, the sequenced DNA and plasmid materials were mixed into a cell culture medium. The previous cell media was replaced by the media containing the DNA, and the genetic material was subsequently transfected into the HEK293 cells.

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Construction of a Chimeric Adeno-associated Virus 2/Human Parvovirus 4 Vector for Gene Therapy | Meriel Doyle â&#x20AC;&#x2122;19

RESULTS AND DISCUSSION:

To produce the chimeric vector, the HEK293 cells were transfected with four plasmidsâ&#x20AC;&#x201C; Cis, Trans, Helper, and our recombinant pshuttle.CMV.PARV4P1.WPRE.SV40pA plasmid. We deleted the AAV capsid gene on the trans plasmid and instead added the fourth recombinant plasmid which would subsequently drive PARV4 capsid protein expression. The wild-type PARV4 genome usually expresses the VP1 and VP2 capsid proteins in a 1:10 ratio. The achievement of this ratio is crucial in creating a functional viral capsid. To achieve this ratio, we devised two different strategies for translation of the capsid proteins:

The first strategy is to use a strong CMV promoter to drive the expression of both VP1 and VP2 while simultaneously weakening the start codon of VP1 so that VP2 will be expressed at a higher level. Normally ATG is the start codon for translation initiation; however, when ATG is replaced with other codons (ACG, CTG, or GTG), translation rates decrease. With VP1 translation reduced, the ratio of VP1 to VP2 should be optimal for capsid formation.

We were able to successfully clone PARV4 VP1 genes under a CMV promoter, while mutating VP1 ATG into CTG, GTG or ACG. During the preliminary experiment, we co-transfected HEK293 cells with the Cis genome plasmid, AAV Rep plasmid, Helper plasmid and one of the mutated PARV4 VP1 plasmids in a 6-well plate. 3 days later, we lysed the cells by freeze and thaw and quantified the DNase resistant Cis genome number by digital droplet PCR. The quantification showed that we were able to obtain about 5E8 genome copies per milliliter (gc/mL) of the DNase resistant genome in each well, with a large variation. This result will be confirmed upon further analysis.

For the second strategy, we will engineer two individual PARV4 plasmids, one encoding only the VP1 gene, and the other encoding only the VP2 gene. We will insert a weak promoter in front of the VP1 fragment, so the expression of the VP1 protein will be reduced. On the other plasmid, we will insert a strong promoter in front of the VP2 fragment to strengthen its transgene expression. This should also create a viral capsid with the proper ratio of VP1:VP2. Different combinations of the plasmids will be used for further vector production testing.

Overall, the preliminary data showed that DNase resistant Cis genomes were produced, suggesting that the chimeric AAV2/PARV4 vector was also successfully produced. Future work will include confirmation and optimization of this chimeric vector system.

ACKNOWLEDGEMENTS:

I would like to thank my mentor, Dr. Weiran Shen, for guiding me throughout my research as well as Shiva Shrestha for teaching me lab techniques and providing me with the materials for my project. I would also like to thank Dr. Jim Wilson for welcoming me in his lab and Dr. Eileen Whiteman for organizing the Gene Therapy Summer Internship Program. Finally, I would like to thank the entire Baldwin Science Department and my biology teacher Mrs. Christie Reed for connecting me to the program.

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HELEN JI â&#x20AC;&#x2122;19 Helen Ji is a senior from Paoli, PA, and has been a student at Baldwin since 9th grade. This past summer, Helen conducted research at the University of Pennsylvania in the Wilson Lab. She is the head of Roman Candle literary magazine and co-head of Cradles to Crayons, captain of varsity crew and a tutor in the Writing Center. In her free time, Helen enjoys volunteering at her local hospital and making baked goods.

Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques Helen Ji â&#x20AC;&#x2122;19 The Baldwin School, Summer Research Program, Gene Therapy Program (GTP) Elizabeth L. Buza, Mentor, Gene Therapy Program (GTP)

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji ’19

INTRODUCTION:

Immunostaining involves using antibodies to label specific antigens (i.e. proteins) of interest in a cell using either fluorescent or nonfluorescent markers, termed immunofluorescence (IF) and immunohistochemistry (IHC) respectively. This process utilizes similar mechanisms of foreign antigen recognition by immune cells employed by the body under normal physiologic conditions, prompting the production of antibodies or other cellular responses to interact specifically and exclusively with the foreign entity.1,2 Mimicking these in vivo mechanisms, immunostaining relies on the specificity of these interactions between antigens and antibodies to bind and label cells in tissue. The antibodies are extracted from various animal species and will bind specifically to the antigens in target cells when applied to tissue of a different species. The specificity of antibodies can be manipulated to bind and label particular cell types based on their unique antigens.3

Direct antigen detection uses a primary antibody labeled with a fluorophore (IF) or color producing enzyme (IHC) to bind to the target antigen (Fig. 1). In indirect detection, a labeled secondary antibody is attached to an unlabeled primary antibody. If the primary antibody has a number of binding sites for the secondary antibody, then numerous secondary antibodies can bind to the primary antibody and produce a larger complex of molecules with a greater number of labeled antibodies, thus exhibiting a stronger signal.4 The color producing labels or fluorophores highlight cells and areas within a cell that have the antigen, which can include the nuclear or plasma membrane.5 Figure 1. IHC/IF uses either direct or indirect antigen detection.

___________________ 1 Maity, Biswanath. Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments. 2013. Accessed August 2, 2018. https://www.ncbi.nlm.nih.gov/pubmed/23317899.

2 “Overview of Immunohistochemistry (IHC).” ThermoFisher Scientific. Accessed August 2, 2018. https://www. thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biologyresource-library/pierce-protein-methods/overview-immunohistochemistry.html.

3 Drew, Clifton P., and Wun-Ju Shieh. Immunohistochemistry. February 13, 2015. Accessed August 4, 2018. https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/ immunohistochemistry.

4 “Direct vs indirect immunofluorescence.” Abcam. Accessed July 30, 2018. https://www.abcam.com/secondaryantibodies/direct-vs-indirect-immunofluorescence.

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19

While this process seems simple and straightforward, there are many instances in which the immunosignal may be inadequate. Though antibodies are specific to antigens, they may bind weakly to other proteins that have similar sequences or structure. When the concentration of either primary or secondary antibody is too high, the antibody could bind to non-target antigens or cells and produce background staining. If the primary or secondary antibody is not specific enough in its binding mechanisms, it will easily bind to other cellular antigens. In high amounts, non-specific binding can result in background staining and mask the signal from the target antigen.6 There are also circumstances in which the level of antigen present in a given tissue may be low, thus also resulting in low signaling. Lastly, some antibodies bind weakly to antigens, producing a weaker signal.

There are a variety of methods that have been developed and/or described to amplify the signal for difficult antigens and/or antibodies. Currently, our lab utilizes a commercially available amplification kit that contains a sensitive biotin-avidin peroxidase system to produce a stronger fluorescent signal with minimal background. Unfortunately, the kit functions only with antibodies in a limited range of animal species (e.g. mouse and rabbit). With the high cost and time constraints created by shipping and manufacturing, the kit can be difficult to obtain. In order to mitigate the disadvantages of the kit, we hypothesize that the level of signal amplification achieved by the commercial kit can be reproduced using two strategies we designed: 1) addition of an intermediate antibody and, 2) a version of biotin-avidin staining technique for amplification.

The intermediary antibody technique we propose utilizes an additional antibody after the primary, followed by the secondary labeled antibody. Because each intermediary antibody has a number of binding sites, the larger molecular complex created will contain a greater number of labels and therefore produce a stronger signal.

Figure 1.5. Intermediary Antibody signal amplification technique

___________________ 6 â&#x20AC;&#x153;Immunohistochemistry (IHC) Guide and Troubleshooting.â&#x20AC;? Accessed August 1, 2018. https://www.mdbioproducts.com/resources/protocols/immunohistochemistry.

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji ’19

The second signal amplification technique we propose utilizes biotin-avidin labeling (Fig. 2). After the primary antibody, a biotinylated secondary antibody is attached. The biotin tag on the secondary antibody facilitates a bond with the avidin substrate. Avidin has an affinity to biotin and four binding sites on each molecule. Because four biotin molecules can bind to a single avidin molecule, a greater number of molecules containing biotin can bind to a protein containing avidin.7 Fluorophores bound to avidin molecules highlight the antigen of choice. This sequence using biotin and avidin can be repeated multiple times, introducing a greater number of fluorochromes to each target antigen, thus providing greater signal amplification.

Figure 2. Avidin-Biotin signal amplification technique.

MATERIALS/METHODS: Preliminary Procedure

Sample brain tissues are extracted from mice and fixed, sectioned, and embedded in paraffin wax. The tissues are sliced to an appropriate thickness (6µm) with a microtome. After labelling each slide, the slides are baked at 65°C for an hour to ensure tissue adhesion to glass. Tissues are deparaffinized and hydrated through xylenes and a graded ethanol series. Slides are then pressure cooked in citrate buffer to expose antigenic sites, allowing antibodies to bind. To increase antibody specificity, a blocking serum is used to prevent unwanted antigen expression. Antibodies can then be applied to the tissue using various techniques to tag the antigen. After staining and amplification techniques are utilized, test slides are coverslipped using Dapi Fluoromount G.

Vectastain Elite ABC Reagent Kit Amplification Technique

After preliminary steps are observed, Olig2 primary antibody (rabbit monoclonal; ab109186; abcam) diluted to 1:500 is applied to slides and incubated. Following the instructions in the Rabbit IgG Kit, the amplifier antibody and Vectofluor reagent are applied successively. Slides are then ready to be coverslipped. ___________________ 7 “Avidin-Biotin Interaction.” ThermoFisher. Accessed July 29, 2018. https://www.thermofisher.com/us/en/home/ life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-proteinmethods/avidin-biotin-interaction.html.

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19

Intermediary Antibody Amplification

Following the preliminary procedures, GFAP as a primary antibody (goat polyclonal; ab53554; abcam) is applied to test slides and diluted to 1:200. After incubation, the rabbit anti-goat IgG intermediary antibody (rabbit polyclonal; ab6714; abcam) at four dilutions (1:200, 1:500, 1:1000, 1:5000) is applied to test slides and incubated at room temperature. For the test slides, a fluorescent antibody (goat polyclonal; 711-025-152; Jackson ImmunoResearch) is applied and incubated. Afterwards, test slides are coverslipped.

Two control slides using GFAP derived from rabbit (rabbit polyclonal; ab7260; abcam) and goat (goat polyclonal; ab53554; abcam) are diluted to 1:1000 and 1:200 respectively. A fluorescent secondary antibody targeting rabbit antibody (goat polyclonal; 711-025-152; Jackson ImmunoResearch) and targeting goat antibody (donkey polyclonal; 705-025-147; Jackson ImmunoResearch) are applied to their respective slides. Slides are then coverslipped following incubation, and observed with microscopy.

Biotin-Avidin Amplification

On one control slide, GFAP primary antibody (chicken polyclonal; ab4674; abcam) is applied at a 1:500 dilution. On one other control slide, the same GFAP is applied at a 1:5000 dilution. Following incubation, a fluorescent secondary antibody targeting chicken antibody (donkey polyclonal; 703025-155; Jackson ImmunoResearch) is applied. After incubation, slides are coverslipped.

To measure the fluorescence from the avidin-fluorescein before amplification through avidinbiotin complexes, a slide is stained using GFAP primary antibody (chicken polyclonal; ab4674; abcam) at 1:5000, followed by a biotinylated chicken IgG antibody (donkey polyclonal; 703-065155; Jackson ImmunoResearch) diluted to 1:500. After avidin fluorescein diluted to 1:200 is applied once, the slide is coverslipped.

On the test slides, GFAP (chicken polyclonal; ab4674; abcam) is applied at 1:5000 and incubated, then biotinylated chicken IgG antibody (donkey polyclonal; 703-065-155; Jackson ImmunoResearch) diluted to 1:500 is applied and incubated. Avidin fluorescein is diluted to 1:200 and applied to all test slides. Biotinylated anti-avidin at three dilutions (1:200, 1:500, 1:1000) is applied to three slides at each dilution, producing nine total slides, and incubated at room temperature. One set of three slides at each concentration is coverslipped. For the other six slides, avidin fluorescein is applied again and incubated. The biotinylated anti-avidin at the same concentrations for each slide as in the previous round is applied again. Following incubation, three slides at each concentration are coverslipped again. On the remaining three slides, avidin fluorescein is applied again, and after incubating, biotinylated anti-avidin is applied at the same concentrations as in the previous two rounds. After incubation, avidin fluorescein is applied to each slide and allowed to incubate. The slides are then coverslipped.

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19

RESULTS:

The commercial kit (Vectastain Elite ABC Reagent Kit) had increased signal amplification compared to our intermediary antibody technique and unamplified positive control.

The antibody Olig2 (rabbit monoclonal; ab109186), was utilized in this first experiment as it is known to have a low immunosignal without amplification. Olig2 identifies oligodendrocytes present throughout the central nervous system. We compared the IF signaling of Olig2 on wildtype mouse brains with the amplification kit, intermediary antibody technique and unamplified positive controls. The results in Figure 3 indicate that the signal amplification of the commercial kit was the strongest. When the exposure was maximized on the intermediary antibody image, a fluorescent signal was still detected indicating that the technique worked; however, less effectively than the commercial kit.

Figure 3. Comparison of fluorescent signal strength of Olig2 on wild-type mouse brain utilizing different amplification techniques.

There was no noticeable signal amplification utilizing our intermediary antibody technique compared to unamplified controls.

The GFAP antibody (goat polyclonal; ab53554) was used to identify astrocytes, which are a type of glial cell present throughout the central nervous system. To test the efficacy of the intermediary antibody technique using antibodies from species not compatible with the commercial amplification kit, the fluorescent signaling of goat GFAP on wild-type mouse brains was compared between unamplified positive controls and intermediary antibody amplification technique. Depicted in Figure 4, the signal strength of the unamplified positive controls was equivalent to the signal strength of the intermediary amplification technique.

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19 Figure 4. Comparison of fluorescent signal strength of GFAP on wild-type mouse brain using intermediary antibody technique

Biotin-Avidin technique resulted in robust signal amplification at the cost of extensive background interference.

The chicken GFAP (chicken polyclonal; ab4674) was utilized to compare unamplified immunolabeling with the biotin-avidin amplification technique. The concentration of the GFAP was decreased tenfold to mimic weaker staining, and the concentration of the biotinylated antiavidin substrate altered to optimize signal amplification (Fig. 5). Comparison of the IF signaling of GFAP between the positive unamplified control and biotin-avidin amplification technique is shown in Figure 5. The signal of the GFAP in the biotin-avidin method was amplified compared to the positive unamplified control; however, significant background staining was present. This nonspecific background staining masks the specific labeling of the astrocytes. These results indicate that the while amplification was achieved with the biotin-avidin method, the positive unamplified control stained most efficiently.

Figure 5. Comparison of fluorescent signal strength of GFAP on wild-type mouse brain using biotin-avidin technique

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19

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Comparison of Immunofluorescence Signal Amplification of a Commercially Available Kit With Other Amplification Techniques | Helen Ji â&#x20AC;&#x2122;19

DISCUSSION:

Due to particular challenges in regards to antibody specificity and level of antigen present in a given tissue, there is need for immunosignal amplification. Currently, our lab utilizes a commercial kit which allows for signal amplification under limited conditions. We hypothesized that our methods of utilizing either an intermediary antibody or an biotin-avidin amplification system would achieve similar results; however, we were unable to optimize these approaches.The commercially available kit provided the strongest signal amplification with the lowest amount of background compared with our two proposed methods of amplification. Our first approach was to test the intermediary antibody method against the commercial kit. The immunofluorescence results indicate that the commercially available kit provided the strongest signal amplification when compared to our intermediary antibody amplification technique (see Fig.4). The intermediary antibody amplification technique did not strengthen the signal of the fluorescent stain when compared to the unamplified positive control (see Fig.4). Our second approach evaluated the effectiveness of a biotin-avidin system against the commercial kit. The biotin-avidin amplification resulted in robust signal amplification; however, this was at the cost of extensive background interference, proving to be less effective than unamplified controls (see Fig.5).

While the commercial kit provided the best signal amplification, there are still limitations, such as species/tissue compatibility and time and money constraints, which dictate a need for an alternative method of signal amplification. Further testing is needed to adequately characterize the ability of the biotin-avidin system for signal amplification. For instance, the background staining may be decreased by using a blocking kit as well as other mechanisms for reducing the amount of nonspecific avidin fluorescein binding, such as altering the concentration of the biotinylated anti-avidin and avidin fluorescein. Further research with other signal amplification techniques could produce more effective methods for future experiments. In conclusion, while our current methods were unsuccessful, there is a demand for alternative immunosignal amplification mechanisms which will be evaluated in the future.

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SHIYU LI â&#x20AC;&#x2122;19 Shiyu is a senior from China and has been a student at Baldwin since 10th grade. She is the head of DECA and Anime, Comics and Games (ACG) club. During the past summers, Shiyu conducted independent research at State Key Laboratory of Medical Neurobiology of Fudan University and interned at Advanced Manufacturing Industrial R&D Center of Tsinghua University. In addition to her interests in science and technology, Shiyu loves playing video games and watching anime.

Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells Shiyu Li â&#x20AC;&#x2122;19 Advisors: Professor Dr. Huawei Li Professor Dr. Yingzi He ENT Institute and Otorhinolaryngology Department of Affiliated Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai.

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

ABSTRACT:

Human hearing disorders are primarily caused by the permanent loss of inner ear hair cells. Testing compounds that modulate hair cell proliferation and regeneration in mammals is necessary but time-consuming and involves complicated processes. The zebrafish is regarded as a suitable model organism for screening drug libraries to study human hearing disorders because of its high fecundity and lateral line mechanosensory hair cells. The zebrafish posterior lateral line, an in vivo screening platform, was utilized in the experiment to identify compounds influencing hair cell death, differentiation, and regeneration after ototoxic drug exposure. Two compounds showed a protective effect from neomycin-induced damage to zebrafish hair cells. Both compounds had no sign of influencing hair cell proliferation and regeneration. The identification of such compounds promoted the understanding of hair cell survival and provides insights in compounds that may participate in the signaling pathway of mechanotransduction. In addition, the regenerative ability of larval zebrafish was assessed using a 6-hour pulse-fix bromodeoxyuridine incorporation protocol to identify the number of proliferating cells in neuromasts. The results proved that cell proliferation in the lateral line mostly happened between 12 to 18 hours following neomycin damage. It also suggested a suitable time period for future studies testing the influence of compounds on hair cell regeneration after ototoxic drug impairment.

KEYWORDS:

hair cell, zebrafish, lateral line, neomycin, compounds

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

TABLE OF CONTENTS 1. Introduction

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2. Methods

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2.1 Animals

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2.2 Drug Treatments

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2.2.1 Hair Cell Development

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2.2.2 Hair Cell Protection

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2.2.3 Hair Cell Regeneration

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2.3 BrdU Incorporation

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2.4 Cell Proliferation Analysis

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2.5 Statistical Analysis

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3. Results

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3.1 Effect of Compounds on Hair Cell Development

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3.2 Effect of Compounds on Hair Cell Protection

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3.3 Effect of Compounds on Hair Cell Regeneration

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3.4 Lateral Line Hair Cell Regeneration after Neomycin-induced Death

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4. Ongoing Studies

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5. Conclusion and Discussion

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

1. INTRODUCTION

Sensory hair cells (HCs) of the inner ear are mechanoreceptors that allow vertebrates to sense sound and orient in space. Excessive noise, ototoxic agents, physical injuries, genetic predispositions, and aging can damage HCs, often leading to permanent hearing and balance disorders.1,2,3 Mammals, including humans, are born with HCs but lack the ability to regenerate lost HCs.

The zebrafish (Danio rerio) is a promising vertebrate model in developmental genetics and biological research. As a model organism, zebrafish are often used to elucidate the mechanisms behind organ development.4 Zebrafish are highly tractable, making them easy to observe and control. Following external fertilization, zebrafish embryos remain transparent until the larvae stage, which makes zebrafish a suitable in vivo model organism. Additionally, breeding zebrafish is less costly than mammalian model organisms because of their small size. The high fecundity also makes zebrafish excellent candidates for drug and genetic screenings.5 Although the zebrafish lacks cochleas, it shares high genetic homology with humans. Previous research shows the genome of zebrafish is approximately 70% parallel to the human genome. As a result, it is feasible to use zebrafish to address issues of human developmental biology and related diseases.

Researchers have sequenced a high-quality genome of zebrafish and provided understandings of its key genomic features. Thus, it is easy to observe mutations and control gene expressions in zebrafish.6 Furthermore, powerful transgenic approaches are widely used and have made zebrafish a model organism fit for large-scale chemical screening and modeling developmental processes4 (Figure 1). For example, the transparent adult zebrafish has been created by researchers to observe and understand stem cells in an in vivo platform.7 Tg(Brn3C:mGFP) transgenic zebrafish display membrane-bound green fluorescent protein (GFP) in hair cells, which makes them ideal tools to observe the hair cells development and survival.

Yamasoba T, et al. (2013) Current concepts in age-related hearing loss: epidemiology and mechanistic pathways. Hear Res 303:30-38.

2 Schacht J, Talaska AE, & Rybak LP (2012) Cisplatin and aminoglycoside antibiotics: hearing loss and its prevention. Anat Rec (Hoboken) 295(11):1837-1850.

3 Konings A, Van Laer L, & Van Camp G (2009) Genetic studies on noise-induced hearing loss: a review. Ear Hear 30(2):151-159.

4 Lieschke GJ & Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8(5):353-367.

5 Whiteley AR, et al. (2011) Population genomics of wild and laboratory zebrafish (Danio rerio). Mol Ecol 20(20):4259-4276.

6 Howe K, et al. (2013) The zebrafish reference genome sequence and its relationship to the human genome.

Nature 496(7446):498-503.

7 Xiao T, Roeser T, Staub W, & Baier H (2005) A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection. Development 132(13):2955-2967.

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

Figure 1. The highlights of zebrafish as model organisms in developmental genetics and biological research.

Zebrafish HCs are structurally and functionally similar to mammalian inner ear HCs.8,9 In addition to HCs in the inner ear, zebrafish have HCs within the lateral line system, which function as detectors of water fluctuations. The lateral line system contains clusters of neuromasts, or centrally-positioned sensory HCs and non-sensory supporting cells, that are arranged along the surface of head and trunk of the fish (Figure 1).10 Zebrafish have two lateral lines: the anterior lateral line surrounding the head and the posterior one along the body. The posterior lateral line is more commonly used in research on hair cells because it consists of neuromasts that are aligned in an orderly manner. Hair cells in the posterior lateral line mature at around 5 dpf (days post fertilization); consequently, experiments on zebrafish larvae could be done in a time-saving manner: they grow much faster compared to the mammalian model organisms. The HCs within the lateral line system are suitable for chemical and genetic screening for several reasons. First, HCs in this system easily take up vital dyes because of their location on the surface of the body and can be visualized in vivo.11 Second, similar to mammalian HCs, zebrafish lateral line HCs are sensitive to ototoxins, such as aminoglycosides antibiotics (including neomycin) and cisplatin.11-13 Whitfield TT (2002) Zebrafish as a model for hearing and deafness. J Neurobiol 53(2):157-171.

Nicolson T (2005) The genetics of hearing and balance in zebrafish. Annu Rev Genet 39:9-22.

10 Thomas ED, Cruz IA, Hailey DW, & Raible DW (2015) There and back again: development and regeneration of the zebrafish lateral line system. Wires Dev Biol 4(1):1-16.

11 Harris JA, et al. (2003) Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol 4(2):219-234.

12 Ou HC, Raible DW, & Rubel EW (2007) Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line.

Hear Res 233(1-2):46-53.

13 Coffin AB, et al. (2010) Chemical screening for hair cell loss and protection in the zebrafish lateral line.

Zebrafish 7(1):3-11.

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

More importantly, unlike mammals, zebrafish have the ability to regenerate HCs within 48 hours of damage.14, 15-18 As nonmammalian vertebrates, zebrafish could regenerate their hair cells by proliferating and differentiating supporting cells. Supporting cells would proliferate after ototoxic drug impairments to replenish the population; then the cells would either differentiate into hair cells or turn into hair cells directly (transdifferentiation). Studying the regenerative mechanisms of zebrafish hair cells is an effective and promising method for developing future treatments for human hearing disorders.

The purpose of the current study is to identify novel compounds that can regulate the survival, development, and regeneration of HCs. Using larval zebrafish lateral line neuromasts as a platform, we undertook a chemical screening, searching for modulators of HCs (Figure 2).

Two compounds were found with the ability to protect HCs against neomycin-induced damage, although they do not affect hair cell development or regeneration. We also characterized HC proliferation in zebrafish lateral line neuromasts after neomycin-induced death and analyzed HC regeneration with respect to time.

Figure 2. Workflow of using zebrafish lateral line system for hair cell studies.

Harris JA, et al. (2003) Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol 4(2):219-234.

15 Lombarte A, Yan HY, Popper AN, Chang JS, & Platt C (1993) Damage and regeneration of hair cell ciliary bundles in a fish ear following treatment with gentamicin. Hear Res 64(2):166-174.

16 Ma EY, Rubel EW, & Raible DW (2008) Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line. J Neurosci 28(9):2261-2273.

17 Williams JA & Holder N (2000) Cell turnover in neuromasts of zebrafish larvae. Hear Res 143(1-2):171-181.

18 Lopez-Schier H & Hudspeth AJ (2006) A two-step mechanism underlies the planar polarization of regenerating sensory hair cells. Proc Natl Acad Sci U S A 103(49):18615-18620.

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li â&#x20AC;&#x2122;19

2. METHODS 2.1 ANIMALS

5 days post-fertilization (dpf ) zebrafish were used to examine the effect of compounds during and following ototoxic drug damage on hair cell protection and regeneration. Zebrafish hair cells in the developmental stage (1-4 dpf ) show resistance to ototoxic drug effects,19 and researchers confirmed that zebrafish hair cells have a mature sensitivity to the toxicity at 5 dpf.19,20 Therefore, the HC regenerative experiments were performed on zebrafish from 5 dpf to 6-7 dpf.

The tests for the effect of compounds on hair cell proliferation was conducted on 3 dpf zebrafish.

Tg(Brn3C:mGFP) transgenic zebrafish larvae were used, because HCs of Brn3c:mGFP animals display membrane-bound green fluorescent protein (GFP) in the lateral line system and inner ear.21 It enables the observation of changes in hair cell number of zebrafish in vivo and makes the lateral line in larvae an ideal platform for drug screening because of the superficial location of the lateral line system and the transparency of zebrafish larvae.

Figure 3. Micrograph of a 3 days post fertilization tg(Brn3C:mGFP) transgenic zebrafish larva. White arrows point to neuromasts P1 to P5 (green dot) on the left side of the larva. Red arrows show neuromasts from the right side of the larva.

Murakami SL, et al. (2003) Developmental differences in susceptibility to neomycin-induced hair cell death in the lateral line neuromasts of zebrafish (Danio rerio). Hearing Res 186(1-2):47-56.

20 Santos F, MacDonald G, Rubel EW, & Raible DW (2006) Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio). Hearing Res 213(1-2):25-33.

21 Xiao T, Roeser T, Staub W, & Baier H (2005) A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection. Development 132(13):2955-2967.

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Using the Zebrafish Lateral Line to Identify Modulators of Mechanosensory Hair Cells | Shiyu Li ’19

2.2 DRUG TREATMENTS

Embryos from Tg(Brn3C:mGFP) heterozygote crosses were collected and examined for GFP expression in the lateral line at 2 dpf, only embryos displaying GFP were used in the tests.

Three separate experiments were conducted to test for the influences of compounds on hair cell development, survival, and regeneration. Compounds dissolved in dimethyl sulfoxide (DMSO) at 10 mM, a concentration that yielded minimal mortality (confirmed by previous trials). Both compound A and compound B are soluble in DMSO. Larvae in control groups were treated only with 10 mM DMSO (≤ 0.2%).

2.2.1 HAIR CELL DEVELOPMENT

To test if the selected compounds influence hair cell development, 3 dpf Tg(Brn3C:mGFP) transgenic zebrafish larvae in different experimental groups were exposed to either compound A (10 or 20 μm) or compound B (2 or 5 μm) for 48 hours. The control group was exposed to only 10 mM DMSO. The counting of GFP-positive HCs was performed on zebrafish fixed in 4% paraformaldehyde (PFA) and rinsed in phosphate-buffered saline (PBS) in 0.1 M PBS at pH 7.2. GFPpositive HCs were counted in four neuromasts per larvae (P1, P2, P3, P4) for 8-10 larvae per group for comprehensive results.

Figure 4. Experimental design for the effect of compounds on hair cell development. Larvae were treated with compounds for 48 h at 3 dpf. Hair cells were counted at 5 dpf. dpf, days post fertilization.

2.2.2 HAIR CELL PROTECTION

To assess the effect of compounds on hair cell survival, 5 dpf Tg(Brn3C:mGFP) transgenic zebrafish larvae were pretreated for 2 hours with compound A (10 or 20 μm) or compound B (2 or 5 μm), followed by 1-hour co-treatment with the compounds and 400 μm neomycin. Larvae were then rinsed with zebrafish embryo medium (EM), placed in EM and anesthetized by MS-222. HCs were then counted by GFP labeling.

Figure 5. Experimental design for the protective effect of compounds from neomycin-induced hair cell death. Larvae were pretreated with compounds for 2 hrs at 5 dpf, followed by 1 hr co-treatment in the compounds and 400 μM neomycin. hpf, hours post fertilization.

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2.2.3 HAIR CELL REGENERATION

To examine how the compounds affect hair cell regeneration, 5 dpf Tg(Brn3C:mGFP) transgenic zebrafish were first treated with 400 μm neomycin to eliminate the hair cells22,23 and then exposed to either compound A or compound B for 24 or 48 hours. In the experiment, zebrafish were rinsed 4 times with EM after neomycin treatment, following the zebrafish laboratory use protocol. Tg(Brn3C:mGFP) transgenic larvae were then placed in 96-well plates and exposed to compound A (10 or 20 μM) or compound B (2 or 5 μM) for 24 or 48 hours recovery periods. The effect of compounds was assessed by the number of GFP labeling hair cells.

Figure 6. The procedure for testing the effect of compounds on HC regeneration. The zebrafish were exposed to compounds for 1 hour prior to the addition of compounds. The number of HCs was counted at 24 hr (6 dpf ) and 48 hrs (7 dpf ) after neomycin treatment.

2.3 BRDU INCORPORATION

To identify the amount of cell proliferation during different 6-hour time periods within the first 48 hours after ototoxic drug impairment, the main HC regeneration period,24 5 dpf larval zebrafish were treated with 400 µM neomycin for 1 hour for hair cells damage, rinsed four times with EM, and exposed to BrdU in EM for different time periods as indicated in Figure 7 at 28.5°C.

Figure 7. Experimental design for early hair cell regeneration. Larvae were treated with neomycin (400 µM) for 1 hour at 5 dpf. Hair cell counting and BrdU-positive cells were analyzed 6, 12, 18, 24 h, and 48 h following initial neomycin exposure. hpa hours post neomycin damage.

Harris JA, et al. (2003) Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol 4(2):219-234.

23 Ma EY, Rubel EW, & Raible DW (2008) Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line. J Neurosci 28(9):2261-2273.

24 Lopez-Schier H & Hudspeth AJ (2006) A two-step mechanism underlies the planar polarization of regenerating sensory hair cells. Proc Natl Acad Sci U S A 103(49):18615-18620.

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2.4 CELL PROLIFERATION ANALYSIS

Cell proliferation was shown by BrdU labeling. The zebrafish larvae were euthanized with MS-222, fixed by PFA and rinsed in 1% PBST,25 and then incubated with Alexa-Cy3 (1:500 dilution in blocking solution; Invitrogen) for 1 hr at 37 °C. DAPI is utilized to show nuclei, and larvae were then rinsed with PBST. Labeled cells were counted in 4 neuromasts per larvae (P1, P2, P3, P4).

Representative figures were examined with a Leica confocal fluorescence microscope (TCS SP8; Leica, Wetzlar, Germany).

2.5 STATISTICAL ANALYSIS

The results were assessed by ANOVA, with GraphPad Prism (see figure legends for details).

Data were analyzed and shown with the p-value. (data with p <0.05 have statistical importance, data with p<0.001 is greatly important.)

3. RESULTS 3.1 HAIR CELL DEVELOPMENT

Neither compound A nor compound B affected the development of hair cell in lateral line neuromasts of zebrafish larvae (Figures 8 and 9). In the test of compound A, control larvae at 5 dpf had 9.75 ± 0.26 hair cells per neuromast. Larvae exposed to compound A have hair cell numbers similar to the number of the control group (Figure 8). In the experiment with the highest compound A concentration tested (20 μM), 5 dpf larvae had 9.58 ± 0.22 hair cells per neuromast.

In compound B experiments, larvae exposed to 5 μM compound B have hair cell numbers similar to the controls (Figure 9). The data suggest that neither compound A nor compound B affects HC development and proliferation.

Namdaran, Parhum, Katherine Reinhart, and Kelly Owens. “Identification of Modulators of Hair Cell Regeneration in the Zebrafish Lateral Line.” J Neurosci., September 7, 2012. 10.1523/ JNEUROSCI.3905-11.2012.

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Figure 8. Compound A did not affect hair cell development in zebrafish larvae. (A-B) Confocal images of neuromasts from zebrafish in the experiment. The zebrafish treated with compound A for 48 hours did not show a great disparity in hair cell number to the control group. (C) Quantification of the GFP+ hair cells in neuromasts. N= 6-10 larvae per treatment.

Data are presented as mean ± SEM

Figure 9. Compound B did not affect hair cell development in zebrafish larvae. (A-B) Confocal images of neuromasts from zebrafish in the experiment. The zebrafish treated with compound B for 48 hours did not show a great disparity in hair cell number to the control group. (C) Quantification of the GFP+ hair cells in neuromasts. N= 6-10 larvae per treatment.

Data are presented as mean ± SEM

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3.2 HAIR CELL PROTECTION

The exposure to 400 μM neomycin caused significant HC loss in the larvae zebrafish (Figure 10). The transgenic zebrafish used in the experiments with green fluorescent neuromasts could be easily observed under fluorescent microscopes. 5 dpf zebrafish were exposed to the compounds for 2 hrs prior to neomycin exposure. The results showed that compound A protected hair cells from neomycin-induced damage. Compared to zebrafish in the control group, whose average number of HC was 1.35 ± 0.19 cells per neuromast, zebrafish with a co-exposure to 20 μM compound A have increased the HC number to 3.8 ± 0.4 (Figure 10C). Similar results were seen in compound B-treated larvae, the treatment of larvae with 5 μM compound B prevented the neomycin-induced hair cell damage with a result of 5.56 ± 0.37 HCs per neuromast. (Figure 11).

Collectively, these data suggest that both compound A and compound B have a protective effect on HCs from the neomycin-induced damage.

Figure 10. The protective effect of compound A from neomycin-induced hair cell damage. (A) Neomycin reduced HC number in zebrafish neuromast. (B) Compound A (20 μM) effectively protected HCs from neomycin damage. (C) Compound A had a protective effect on HCs in the zebrafish lateral line neuromasts. **p < 0.01.

Data are presented as mean ± SEM.

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Figure 11. The protective effect of compound B from neomycin-induced hair cell damage. (A) Neomycin reduced HC number in zebrafish neuromast. (B) Compound B (20 μM) effectively protected HCs from neomycin damage. (C) Compound B had a protective effect on HCs in the zebrafish lateral line neuromasts. **p < 0.01.

Data are presented as mean ± SEM.

3.3 HAIR CELL REGENERATION

Exposure to different compounds may affect HC regeneration. We examined the effect of compound A and compound B after the neomycin-induced damage. Larvae were treated with 400 μM neomycin, then exposed to compounds, or DMSO (in the control group). The results showed that compound A had no effect on HC regeneration (Figure 12). Regeneration started within the first 24 hours after neomycin damage and completed around 48 hrs post damage, whether with or without the presence of compound A. Similar results were shown in compound B experiments: with the exposure to 5 μM compound B, the regeneration process was not different from the control group (Figure 13). In short, the data suggest that neither compound A nor compound B affects lateral line HC regeneration.

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Figure 12. Compound A treatment didn’t affect hair cell regeneration. Confocal images (A-D) showed that the exposure to compound A did not result in significant changes in the number of HCs compared to the control group.

Data are presented as mean ± SEM

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Figure 13. Compound B treatment didn’t affect hair cell regeneration. Confocal images (AD) showed that the exposure to compound B did not result in significant changes in the number of HCs compared to the control group. Data are presented as mean ± SEM

3.4 REGENERATION WITH RESPECT TO TIME

Previous studies report that hair cell regeneration is evident at 24 h post-neomycin treatment and is largely complete after 48 h.26 We examined neuromast HCs at 24 and 48 hours after neomycin exposure, using GFP labeling to show the changes in hair cell populations in lateral line neuromasts of zebrafish larvae. Our results showed that most of the HCs were damaged immediately after neomycin treatment. When larvae were given 48 hours to recover, HCs regenerated and looked healthy, as illustrated in Figure 14 A and B. To further explore the proliferative events following neomycin impairment, the amount of cell proliferation in the lateral line was observed at 24 and 48 hours following neomycin treatment using BrdU incorporation. We calculated the BrdU index to determine the level of neuromast cell proliferation, which is the ratio of BrdU positive cells to all cells in the neuromast. 24 h after neomycin treatment, the BrdU index was about 39%. At 48 h post neomycin treatment, the BrdU index was 46% (Figure 14). This rate of proliferation was similar to that in previous research regarding HC regeneration in larval zebrafish; however, it remained unknown in which time period the major recovery happened in the first 24 hours following neomycin damage. We examined the regenerative ability of larval zebrafish using a 6 h pulse-fix BrdU incorporation protocol to identify the number of S-phase cells in neuromasts.

Ma EY, Rubel EW, & Raible DW (2008) Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line. J Neurosci 28(9):2261-2273.

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Larvae were treated with neomycin for 1 hr and then allowed to regenerate hair cells for 6, 12, 18 or 24 h after initial exposure. BrdU labeling showed the number of cells in S-phase. Figures 15A-D show representative neuromasts from animal 6, 12, 18 and 24 h after 400 μM neomycin treatment. Figure 15E presents the results. Several cells labeled with BrdU were seen in 0-6 h and 6-12 h groups. A significant increase in the amount of labeled HC was observed in between 12-18 h following neomycin exposure. The results indicated that cell proliferation mostly occurs between 12 and 18 h after neomycin damage.

Figure 14. Hair Cell regeneration after neomycin-induced death in lateral line neuromasts.

(A-B) Representative neuromasts were exposed to 400 μM neomycin, then pulsed with BrdU for 24 h (A) or 48 h (B), and immediately fixed. This neuromast contained proliferating cells (BrdU+ cells). (C) Quantification of the ratio of BrdU+ cells/ all neuromast cells in larvae at 24 and 48 hours after neomycin exposure.

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Figure 15. Cell proliferation mostly occurs between 12 and 18 h after neomycin damage. (A-D) Representative neuromasts were exposed to 400 μM neomycin, pulsed with BrdU during 0-6 h (A), 6-12 h (B), 12-18 h (C) or 18-24 h (D) after neomycin-induced damage, and then immediately fixed. (E) Quantification of the BrdU+ cells in neuromast for each experimental condition. Most of the regeneration of HCs in neuromasts happened during 12-18 hours period. Data are presented as mean ± SEM **p<0.001.

4. ONGOING STUDIES

We consider the studies conducted to date on HC proliferation, protection, and regeneration as proofs for the principle that the zebrafish lateral line is a preferred tool to discover novel compounds that may reach clinical use. In addition to compounds identified in this report, more compounds from drug libraries are being examined for their effects on hair cell development, protection, and regeneration.

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5. CONCLUSION AND DISCUSSION

The posterior lateral line system consists of mechanosensory hair cells highly similar to those in the human inner ear. Hair cells in the neuromasts were used in the experiments to identify compounds with protective effects from aminoglycoside neomycin-induced HC death.

5 dpf zebrafish larvae were used for neomycin damage because hair cells of larvae weren’t fully developed until 5 dpf. HCs in the lateral line usually start to grow at 2 dpf, and developing hair cells show resistance to neomycin effects. 5 dpf zebrafish started to show sensitivity for ototoxic drugs.27,28 Larvae zebrafish were separated into plates, exposed to compounds for 2 hours, and then to both neomycin and the compounds for an hour. Ten compounds were screened and two of them (compound A and compound B) were identified as exhibiting a protective effect on hair cells from the neomycin damage. The identified compounds in the experiments are candidate protectants that could someday reach clinical use. The effect of compound A and B on HC development and regeneration was also tested. The results showed that both compounds did not affect any of the processes. Future studies are needed to test the effect of the compounds on mammalian inner ear hair cells.

After the insults of neomycin, larvae zebrafish regenerate their HCs within 48 hours after the damage, by proliferating the supporting cells and differentiating them into HCs,29,30 while the time period during which most regeneration occur remained unknown. By using a 6 h pulsefix BrdU incorporation protocol, the quantity of cell proliferation was assessed for each 6-hour period in the first 48 hours after neomycin treatment. The HCs in neuromasts were found to have the maximum regenerative ability between 12 and 18 h after neomycin damage. The result is a valuable reference for future research on the regeneration of zebrafish hair cells, as it indicates the time frame for HC recovery. Furthermore, we anticipate that we can utilize the regenerative ability of zebrafish hair cells and the suitable time course to create a useful method for drug screening in the future to identify compounds that are the modulators of HCs.

Zebrafish is a useful model organism for the identification of drugs that applicable to human hearing disorders. The compounds that affect hair cell proliferation and regeneration also provide insights into the auditory signaling pathways. A further understanding of hair cell development, protection, and regeneration will enable researchers to design new medications correspondingly and to optimize the effects.

ACKNOWLEDGMENTS

I am grateful for the support and supervision that Professor Huawei Li and Professor Yingzi He at ENT Institute and Otorhinolaryngology Department of Affiliated Eye and ENT Hospital of Fudan University have offered. They inspire my research and encourage me to explore the field of biological sciences. Their research on zebrafish hair cells is fabulous!

Murakami SL, et al. (2003) Developmental differences in susceptibility to neomycin-induced hair cell death in the lateral line neuromasts of zebrafish (Danio rerio). Hearing Res 186(1-2):47-56.

28 Santos F, MacDonald G, Rubel EW, & Raible DW (2006) Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio). Hearing Res 213(1-2):25-33.

29 Harris JA, et al. (2003) Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol 4(2):219-234.

30 Ma EY, Rubel EW, & Raible DW (2008) Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line. J Neurosci 28(9):2261-2273.

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MINGXIN SHI ’19 Mingxin came to Baldwin in 2016 and currently resides in Bryn Mawr, PA. She is the co-head of DECA, BETA and is a staff writer for the student newspaper, The Hourglass.

Preparation of Porous and Recyclable PVA-TiO2 Hybrid Hydrogel Mingxin Shi ’19 Gen Li PhD The Baldwin School Institute of Chemistry Chinese Academy of Sciences

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Preparation of Porous and Recyclable PVA-TiO2 Hybrid Hydrogel | Mingxin Shi ’19

ABSTRACT

Nano TiO2, one of the most effective photocatalysts, has extensive use in fields such as air purification, sewage treatment, water splitting, reduction of CO2, and solar cells. Nowadays, the most promising method to recycle nano TiO2 during the photocatalysis is to immobilize the TiO2 onto a matrix, such as polyvinyl alcohol (PVA). However, due to the slow water permeability of PVA after cross-linking, the pollutants could not efficiently contact the nano TiO2 photocatalyst particles. To overcome this problem, we dispersed calcium carbonate particles into a PVA-TiO2 mixture and coated glass with the mixture. PVA-TiO2-CaCO3 films were obtained by drying.

Through thermal treatment, we obtained the cross-linked PVA-TiO2-CaCO3 films. Finally, the calcium carbonate in the film was dissolved by hydrochloric acid, and the porous PVA-TiO2 composite photocatalyst was obtained. The results show the addition of CaCO3 has no obvious effect on PVA cross-linking and that a large number of cavities have been generated on the surface and inside of porous PVA-TiO2 hybrid hydrogel film. The size of the holes is about 5-15μm, which is consistent with that of CaCO3.The photocatalytic rate constant of porous PVA-TiO2 hybrid hydrogel film is 2.49 times higher than that of nonporous PVA-TiO2 hybrid hydrogel film.

KEY WORDS

Hybrid Hydrogel, Photocatalysis, TiO2, PVA

I. INTRODUCTION Titanium dioxide, TiO2, one of the white pigments, is widely used in paints, plastics, and cosmetics. Different from silicon dioxide, calcium carbonate, clay, and other materials, TiO2 has a unique photocatalytic function. The photocatalytic principle of TiO2 is shown in Fig.1.1 When TiO2 is exposed to sunlight, especially ultraviolet rays, its valance electrons move to the conduction band, resulting in a free electron-hole pair. The free electron-hole pair has strong oxidation-reduction capability and can react with oxygen and water in the air to produce reactive oxygen and hydroxyl radicals. When benzene, toluene, formaldehyde, bacteria, viruses and other pollutants adsorb on the surface of TiO2, they are going to combine with free electrons or holes, have an oxidation reduction reaction, and be decomposed into carbon dioxide, water, etc. Therefore, TiO2 is one of the photocatalysts with huge potential, and has been widely applied to air purification, sewage treatment, water splitting, reduction of CO2 and solar cells.2 3 4 5 J. Mo, Y. Zhang, Q. Xu, J.J. Lamson, R. Zhao, Photocatalytic purification of volatile organic compounds in indoor air: A literature review, Atmospheric Environment, 43 (2009) 2229-2246.

2 H. Zhang, X. Lv, Y. Li, Y. Wang, J. Li, P25-Graphene Composite as a High Performance Photocatalyst, ACS Nano, 4 (2010) 380-386.

3 Dhandole L K, Mahadik M A, Kim S G, et al. Boosting photocatalytic performance of inactive rutile TiO2 nanorods under solar light irradiation: Synergistic effect of acid treatment and metal oxide co-catalysts[J]. Acs Appl Mater Interfaces, 9(2017) 23602–23613.

4 Kim S-G, Dhandole LK, Lim J-M, Chae W-S, Chung H-S, Oh B-T, et al. Facile synthesis of ternary TiO2 NP/ Rh & Sb-codoped TiO2 NR/titanate NT composites photocatalyst: Simultaneous removals of Cd2+ ions and Orange (II) dye under visible light irradiation (λ≥420nm). Applied Catalysis B: Environmental, 224(2018) 791803.

5 Sarker S, Mukherjee B, Crone E, et al. Development of a highly efficient 1D/0D TiO2 nanotube/n-CdTe photoanode: single-step attachment, coverage, and size control by a solvothermal approach[J]. Journal of Materials Chemistry A, 2(2014) 4890-4893.

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With the continuous decrease of TiO2 particle size, the specific surface area of TiO2 increases continuously, and its photocatalytic activity also increases accordingly6. Therefore, all TiO2 used for photocatalysis is nano TiO2. However, it is difficult to separate and recycle nanometer TiO2 particles in the process of application. If not recycled, the loss of nano TiO2 particles to the environment will be threatening to the ecosystem and human health. To separate P25 from the water, researchers usually have to use a centrifuge (10000r/min, 5min) or filter (0.22um ultrafiltration membrane, 3MPa). The process is complicated and expensive. In order to overcome this application bottleneck of nano TiO2, the most effective method is to load nano TiO2 particles in large-size solid carriers to prepare the nano TiO2 photocatalyst. The carriers can be categorised into inorganic carriers and organic carriers. The former mainly includes glass beads, ceramics, clay and aluminium foil7, while the latter mainly includes polyethylene terephthalate, polypropylene, cellulose and activated carbon.8 Shang et al.9 coated the TiO2 sol on the surface of stainless steel wire mesh and obtained the high specific surface area and high catalytic activity loaded TiO2 by calcine. The results showed that the loaded TiO2 had good photocatalytic effect on formaldehyde. Shiva et al. 10 used alkali and surfactant to treat polyester fabrics to make the polyester fiber surface have some holes and hydrophilic groups, then immersed the fabric into the dispersion liquid of nanometer TiO2, took it out after ultrasonic treatment, and carried out high temperature treatment. The results demonstrated that the fabric has good catalytic effect on methylene blue.

Polyvinyl alcohol (PVA) is a kind of water-soluble polymer material that contains many hydroxyl groups. Due to its excellent film-forming, water solubility, mechanical properties and biodegradability, it has been widely applied in the fiber, film, emulsifying agent, binder, and other fields. Pure PVA film has no obvious absorption of UV and visible light and is very suitable to support nanometer TiO2 photocatalyst11 particles. However, due to the slow water permeability of PVA after cross-linking, the pollutants cannot efficiently contact the nano TiO2 photocatalyst.

Therefore, the rate of photocatalysis of nanometer TiO2 photocatalyst will decrease when loaded into the PVA film. To solve this problem, Song et al.12 coated the PVA/TiO2 composite photocatalyst Sun Fengyu, Wu Ming , Li Wenzhao, Li Xinyong, Gu Wanzhen, Wang Fudong. Relationship between crystallite size and photocatalytic activity of titannium dioxide. Chinese Journal of Catalysis.19(1998) 229-233.

7 N. Daneshvar, D. Salari, A. Niaei, M.H. Rasoulifard, A.R. Khataee, Immobilization of TiO2 nanopowder on glass beads for the photocatalytic decolorization of an azo dye C.I. Direct Red 23, Environmental Letters, 40 (2005) 1605-1617.

8 S. Matsuzawa, C. Maneerat, Y. Hayata, T. Hirakawa, N. Negishi, T. Sano, Immobilization of TiO2 nanoparticles on polymeric substrates by using electrostatic interaction in the aqueous phase, Applied Catalysis B: Environmental, 83 (2008) 39-45.

9 J. Shang, W. Li, Y. Zhu, Structure and photocatalytic characteristics of TiO2 film photocatalyst coated on stainless steel webnet, Journal of Molecular Catalysis A: Chemical, 202 (2003) 187-195.

10 S. Hashemizad, M. Montazer, A. Rashidi, Influence of the surface hydrolysis on the functionality of poly(ethylene terephthalate) fabric treated with nanotitanium dioxide, Journal of Applied Polymer Science, 125 (2012) 1176-1184.

11 P. Lei, F. Wang, X. Gao, Y. Ding, S. Zhang, J. Zhao, S. Liu, M. Yang, Immobilization of TiO2 nanoparticles in polymeric substrates by chemical bonding for multi-cycle photodegradation of organic pollutants, Journal of hazardous materials, 227-228 (2012) 185-194.

12 Y. Song, J. Zhang, H. Yang, S. Xu, L. Jiang, Y. Dan, Preparation and visible light-induced photo-catalytic activity of H-PVA/TiO2 composite loaded on glass via solâ&#x20AC;&#x201C;gel method, Applied Surface Science, 292 (2014) 978-985.

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on a glass plate and tried to prepare thinner composite film. Zhang et al.13 coated the PVA/ TiO2 composite photocatalyst on honeycomb ceramics in an attempt to prepare a more active photocatalyst. Nguyen et al.14 prepared a porous PVA/TiO2 composite photocatalyst by freezedrying and improved the photocatalytic activity of the catalyst. Lee et al.15 prepared nano PVA/ TiO2 composite fiber membranes by electrostatic spinning, and found that the photocatalytic activity of the composite catalyst was inversely proportional to the diameter of the fiber. However, there are still many challenges in developing a cheap and environmentally friendly method for the preparation of efficient and recyclable TiO2 composite photocatalyst.

Fig. 1. A schematic diagram of the catalytic degradation of pollutants by TiO2 under UV radiation

As far as we know, no one has prepared a porous PVA-TiO2 composite photocatalyst by etching calcium carbonate from hydrochloric acid. Here, we dispersed calcium carbonate particles into a PVA-TiO2 mixture and then filmed the glass. Through thermal treatment, we obtained the cross-linked PVA-TiO2-CaCO3 films. Finally, the calcium carbonate in the film was dissolved by hydrochloric acid, and a porous PVA-TiO2 composite photocatalyst was obtained. The morphology, structure, and photocatalytic performance of the composite catalyst were also characterised.

II. EXPERIMENT A. MATERIALS

PVA (average degree of polymerisation 1750±50): Beijing Yili chemical co. LTD. Calcium carbonate (AR): Sinopharm chemical reagent co. LTD. Hydrochloric acid (AR): Beijing Chemical Plant. P25 (about 20% rutile and 80% anatase with an average particle size of 21nm): Germany Evonik Company. Methyl Orange (model pollutant): Zhejiang Yongjia Chemical co.

B. PROCEDURE

Preparation of PVA Solution: add 15g PVA to a 250ml beaker, and then add 185ml of deionised water. Agitate mechanically (300r/min). After swelling up for an hour, raise the temperature to J. Zhang, H. Yang, L. Jiang, Y. Dan, Enhanced photo-catalytic activity of the composite of TiO2 and conjugated derivative of polyvinyl alcohol immobilized on cordierite under visible light irradiation, Journal of Energy Chemistry, 25 (2016) 55-61.

14 Y. Song, J. Zhang, L. Yang, S. Cao, H. Yang, J. Zhang, L. Jiang, Y. Dan, P. Le Rendu, T.P. Nguyen, Photocatalytic activity of TiO2 based composite films by porous conjugated polymer coating of nanoparticles, Materials Science in Semiconductor Processing, 42 (2016) 54-57.

15 N.T.B. Linh, K.-H. Lee, B.-T. Lee, Fabrication of photocatalytic PVA–TiO2 nano-fibrous hybrid membrane using the electro-spinning method, Journal of Materials Science, 46 (2011) 5615-5620.

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95oC. Dissolve for three hours and get a transparent and homogeneous PVA solution with a solubility of 7.5%.

Add 300mg TiO2 to a 150ml beaker, add 15ml deionised water, disperse by ultrasonic for 2min, then add 40g PVA solution (7.5% solubility), and stir for 15 min to get PVA-TiO2 solution. Add 1.5g CaCO3 and 5ml deionised water to a 10ml centrifuge tube. After ultrasonic dispersion for 2min, the solution was added to PVA-TiO2 solution, and the PVA-TiO2-CaCO3 solution was obtained after stirring for half an hour. Pour the dispersing liquid onto the glass plate and use the film laying machine to lay a 1mm thick film. The thickness of the film after natural drying was about 45μm. Cover the dried film with two pieces of A4 paper to prevent curling and place inside a 140o oven. Take it out after cross linking for 2 hours in the vacuum state, and get the cross linked PVA-TiO2-CaCO3 film. Cut a certain amount of cross linked film and put in 300ml 1% HCl solution.

Lots of bubbles were produced on the surface. After soaking for half an hour, clean the film with deionised water for 6 times and get a porous PVA-TiO2 hydrogel film.

C. EXPERIMENTAL INSTRUMENTS

A scanning Electron Microscope (SEM, SU8020) was used to observe the morphology of the sample, and the acceleration voltage was 15KV.

The sample’s Fourier Transform Infrared Spectroscopy (FTIR) analysis used Thermal Fisher company’s Nicolet Avatar 6700 reflection accessory of FTIR Nicolet Smart Orbit Accessory (Thermo Fisher Scientific). The wave number rage was 4000-650cm-1. The resolution was 4cm-1 and scanned 32 times.

UV-Vis Diffuse reflection spectrum (UV-Vis DRS) was measured by Shimadzu Company’s UV2600 ultraviolet spectrophotometer, equipped with integral ball attachments. The test resolution was set to 1nm, using Barium Sulfate for baseline correction, and the scanning rage was 200800nm.

The Thermo-gravimetric analysis under air(TGA-air) used Perkin-Elmer TGA-7 series thermal analysis system. The temperature range was 100oC to 700oC. The heating rate was 20oC/min, and the air velocity was 20ml/min.

D. PHOTOCATALYTIC DEGRADATION

Methyl Orange (MO) was selected as the target pollutant to evaluate the photocatalytic performance of the new UV light catalyst. The initial concentration of methyl orange was 15mg/L. The UV light catalytic experiment was carried out in the photochemical reactor produced by Beijing Zhongjiao Jinyuan Company. The light source was a 500W xenon lamp, and the ultraviolet light of 365±15nm was obtained by the addition of the filter. Before illumination, measured a sample containing 20mg TiO2 nanoparticles and put into a beaker containing 40ml methyl orange solution. Two hours in darkness was allowed to achieve the adsorption-desorption equilibrium.

After illumination, the absorbance of 5ml filtrate was measured at regular intervals of every half an hour. The photocatalytic mechanism of Methyl Orange by TiO2 is shown in the figure below16. The absorbance of MO solution at the maximum absorption wavelength of 465 nm was measured by the ultraviolet visible absorption spectrometer (Lovibond, ET99731). The measurement was Bianco P A, Basso A, Baiocchi C, et al. Analytical control of photocatalytic treatments: degradation of a sulfonated azo dye. Isozymes, 378(1975) 297-312.

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repeated three times. The mean value was calculated. Ct/C0 (=At/A0) was used to describe the degradation degree of MO, and the Ct/C0-t curve was drawn.

Fig. 2. Possible degradation pathway for MO during photocatalysis

III. RESULTS AND DISCUSSIONS A. PICTURES

Fig.3. shows the pictures of pure PVA, PVA-TiO2, and PVA-TiO2-CaCO3 films. Picture A shows that the pure PVA film is transparent, so it is very suitable for TiO2 loading. After adding TiO2, the film turns out to be white with a slightly reflective surface. PVA-TiO2-CaCO3 film is also white, but the surface reflect light significantly diminishes, showing that its diffuse reflection increases, and its surface is rough comparing to that of PVA-TiO2.

B. FTIR GRAPH ANALYSIS OF CACO3, PVA, PVA-TIO2, AND PVA-TIO2CACO3 FILMS

Fig. 4. is the FTIR curves of CaCO3, PVA, PVA-TiO2, and PVA-TiO2-CaCO3 thin films. In Fig.4., the infrared spectrum of CaCO3 mainly has two absorption peaks, 1377cm-1 and 864cm-1, respectively, corresponding to the symmetric stretching vibration of C-O and the deformation vibration peak of external CO surface. Pure PVA in 3276cm-1, 2920cm-1, 1655cm-1, 1427cm-1, and 1080cm-1 has obvious absorption peaks. Those absorption peaks correspond to the OH stretching vibration, CH2 stretching vibration, OH bending vibration, CH2 bending vibration and C-O stretching vibration, respectively. The infrared absorption peak of PVA-TiO2 after cross-linking is consistent with that of pure PVA, indicating that the addition of TiO2 has no obvious effect on the cross linking of PVA. It is worth noting that PVA-TiO2-CaCO3 film has a wide absorption peak in the 1400cm-1, and the absorption peak becomes sharp in 874cm-1, which is mainly caused by the addition of CaCO3.

After comparing the infrared spectroscopy of PVA-TiO2-CaCO3 and PVA, it can be shown that the addition of CaCO3 has no effect on PVA cross-linking. This report does not include the analysis

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on the intensity change of different data because under FTIR’s reflection mode, the intensity of peaks is related to the sample pressure. Additionally, combining the result of TGA curves, the initial degradation temperature of PVA is higher than 250oC, indicating that the thermal treatment at 140oC by P25 and CaCO3 has no effect on PVA.

Fig. 3. Pictures of pure PVA, PVA-TiO2, and PVA-TiO2-CaCO3 films

Fig. 4. FTIR graph of CaCO3, PVA, PVA-TiO2, and PVA-TiO2-CaCO3 films

C. TGA CURVE ANALYSIS OF PURE PVA, PVA-TIO2, AND PVA-TIO2CACO3 FILMS

Fig. 5. is the TGA curves of pure PVA, PVA-TiO2, and PVA-TiO2-CaCO3 films. The figure shows that the pure PVA film has good heat resistance, with only about 0.7% of weight loss before 250oC, which is due to PVA intermolecular cross linking dehydration. PVA thermal degradation mainly consisted of three stages. The first phase is between 250-350oC. The process is mainly the hydroxyl removal and the chain break of part of the PVA molecular chain. The second phase is 350-450oC. The process is mainly the oxidisation degradation of PVA molecular chain and carbide of the molecular chain. The third phase is 450-570oC. The process is the oxidation and degradation of PVA carbide products. After adding TiO2, the initial pyrolysis temperature of PVA was slightly advanced, which may be caused by the thermal catalysis of TiO2. PVA-TiO2-CaCO3 film’s initial thermal decomposition temperature also moves in advance compared to that of pure PVA, but based on the curve of the process of degradation, TiO2 and CaCO3 only slightly reduced the heat resistance of PVA, but have not effect on the degradation of PVA. In addition, pure PVA’s thermal weight loss above 600oC is 100%, proving that it can decompose completely, so the residual weights of PVATiO2 and PVA-TiO2-CaCO3 films under 600oC are the inorganic contents, about 10% and 40% THE BALDWIN REVIEW 2018

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respectively, demonstrating that PVA-TiO2 film contains 10% of TiO2 and PVA-TiO2-CaCO3 film contains 10% TiO2 and 30% CaCO3.

D. SEM ANALYSIS OF PURE TIO2

Fig. 6. contains the SEM photos of pure TiO2. The figure shows that the pure TiO2 has some agglomerates, with size of around 5-15μm. Based on the high power photos, the aggregates are composed of nano TiO2 particles with even smaller size. The primary size of TiO2 particles is less than 50nm and is uniform, proving that nanoscale TiO2 is used.

Fig. 5. TGA curves of pure PVA, PVA-TiO2, and PVA-TiO2-CaCO3 films (air, 20oC/min)

Fig. 6. SEM photos of pure TiO2

E. SEM ANALYSIS OF CACO3, PVA-TIO2, AND PVA-TIO2-CACO3 FILMS

Fig.7. shows the SEM photos of CaCO3, PVA-TiO2, and PVA-TiO2-CaCO3 films. As can be seen from Picture A, CaCO3 particles are mainly micron particles with uneven size. Most of them are 5-15μm irregular particles, and a large number of CaCO3 particles with a size of 1-5μm also exist in the system. Based on Picture B, the surface of PVA-TiO2 film surface is smooth without holes, which is not conducive to the entry and exit of pollutant solution. In addition, a large number of white areas can be seen on the surface of PVA-TiO2 film, which may be the aggregates of TiO2. The size of these white areas is about 10μm, indicating that TiO2 has agglomeration in PVA. As shown in Picture C, after etching, PVA-TiO2-CaCO3 film surface appears to have a large number of cavities.

The size of the holes is about 5-20μm, which is consistent with that of CaCO3, showing that CaCO3 in the film is dissolved and a porous PVA film is successfully obtained. Moreover, it can be seen

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from Picture D that the thickness of PVA-TiO2-CaCO3 film is about 45Îźm. There are also a large number of holes in in the inner part of PVA-TiO2-CaCO3 film, proving that the method of etching CaCO3 can successfully prepare porous PVA hydrogels. At this point, the SEM pictures prove that there are large number of cavities, which sizes are the same as those of CaCO3, as shown in Picture A and C. We plan to carry out AFM test and water permeability test in the next step.

F. ANALYSIS OF THE CHANGE OF METHYL ORANGE CONCENTRATION

Methyl Orange is used as a model pollutant to characterise the catalytic activity of nonporous and porous PVA-TiO2 hybrid hydrogels under UV lights. Prior to the experiment, pure methyl orange solution was placed under UV light for two hours, and its concentration remained unchanged. As shown in Fig. 7, when added to our hydrogels, the concentration of methyl orange decreases under the light, indicating that both nonporous and porous PVA-TiO2 hybrid hydrogels can catalyse the degradation of methyl orange. Nonporous PVA-TiO2 hybrid film degrades about 54% of methyl orange under UV light for 2 hours, while porous PVA-TiO2-CaCO3 film degrades about 86% of methyl orange, showing that the porosity of the film allows pollutants to contact with the film at a faster rate and increases the contact of TiO2 and pollutants, and then improves the photocatalytic performance of the film.

Fig. 7. SEM photos of CaCO3, PVA-TiO2, and PVA-TiO2-CaCO3 films

Fig. 8. The change curves of Methyl orange concentration catalysed by UV light

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Fig. 9. Photocatalytic degradation of methyl orange reaction rate fitting curve

The rate of degradation process of methyl orange catalysed by PVA-TiO2-CaCO3 film decreases slightly as the the concentration of methyl orange decreases, indicating that the photocatalytic reaction accords with first-order reaction. In Fig. 8, showing ultraviolet illumination time t based on ln(Ct/C0) with a linear fit, the slope of the linear fit is the photocatalytic reaction rate constant. PVATiO2 and PVA-TiO2-CaCO3 film photocatalytic rate constants are 0.39h-1 and 0.97h-1, respectively. The higher the rate constant, the higher the photocatalytic efficiency, indicating that the photocatalytic rate of porous PVA-TiO2-CaCO3 film is 2.49 times than that of nonporous PVA-TiO2 film.

G. ANALYSIS OF THE RECYCLABILITY OF POROUS PVA-TIO2-CACO3 FILM

In Fig. 10, it shows that the degradation curve of methyl orange photo-catalysed by porous PVATiO2-CaCO3 film fits the first-order kinetics, so the photocatalytic effect of PVA-TiO2-CaCO3 film is pretty stable. However, figure 10 also demonstrates that the hybrid film’s photocatalytic activity slightly decreases after testing for five times. This was probably because the degradation products of methyl orange stay in the hybrid film or get absorbed on the surface of TiO2. Moreover, under UV light irradiation, TiO2 might catalyse the matrix of PVA, leading to the slight decrease of TiO2’s photocatalytic activity. In conclusion, after repeating the cycle of photocatalysis for 5 times, the hybrid film still has photocatalytic activity, with only a slight decrease. In later experiment, we will try to prepare more stable film.

Fig. 10. Cycle photocatalytic performance test of PVA-TiO2-CaCO3 under UV light irradiation H10

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IV. CONCLUSION

Porous PVA-TiO2 hybrid hydrogel film was successfully prepared by etching CaCO3 with hydrochloric acid. This method is safe, green, simple, and easy to be prepared in large quantities.

TGA shows that the addition of CaCO3 volume is about 30%. FTIR shows the addition of CaCO3 has no obvious effect on PVA cross-linking. SEM shows that a large number of holes has been generated on the surface and inside of porous PVA-TiO2 hybrid hydrogel film. The size of the holes is about 5-15Îźm, which is consistent with that of CaCO3.

Porous PVA-TiO2 hybrid hydrogel film has significantly high photocatalytic efficiency than that of nonporous hybrid hydrogel film. The photocatalytic rate constant of porous PVA-TiO2 hybrid hydrogel film is 2.49 times higher than that of nonporous PVA-TiO2 hybrid hydrogel film since the porosity increases the contact area and rate of TiO2 and pollutants. The film is recyclable and its photocatalytic rate does not decrease when repeating the experiment.

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ANJALI SUKHAVASI ’19 Anjali Sukhavasi is a senior from Glen Mills, PA, and has attended Baldwin since 9th grade. She is head of the Mock Trial Club and Asian Students’ Association. Anjali also participates in Lamplighters and Model UN. In her free time, Anjali enjoys dancing and reading.

Decreasing Pancreatic Ductal Adenocarcinoma Cell Viability

by Increased ROS Levels Through IDH1 Inhibition

By Anjali Sukhavasi ’19 Thomas Jefferson Hospital, Department of Surgery

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Decreasing Pancreatic Ductal Adenocarcinoma Cell Viability by Increased ROS Levels Through IDH1 Inhibition | Anjali Sukhavasi ’19

INTRODUCTION

The pancreas is an organ in the gastrointestinal, or digestive tract. It is 6-10 inches long and 2 inches wide, situated in the upper abdomen behind the stomach, next to the small intestine. Its primary function is producing digestive enzymes, such as insulin, that are released through the main pancreatic duct into the duodenum, a portion of the small intestine, as seen in Figure 1,12 These pancreatic enzymes are vital to blood sugar regulation, also referred to as glucose homeostasis.

Figure 1

PANCREATIC CANCER

Pancreatic ductal adenocarcinoma, hereafter referred to as PDAC, is a cancer of the exocrine cells that line the main pancreatic duct, as seen in Figure 2.3 It is the 3rd most frequent cause of cancer-related deaths in the United States and is expected to rise to the 2nd most common cause of cancer-related deaths within the next decade. It is a deadly disease that is only curable for patients with localized, contained tumors, as they could undergo surgery to completely remove cancerous cells.4 However, to this day, there is no effective screening test for PDAC. Common symptoms associated with the disease include fatigue, diabetes, and weight loss, which are common in many other diseases, as well. There is usually little to no pain or Figure 2 discomfort in the area that could confirm the presence Jordan Winter, Jonathan Brody, Ross Abrams, James Posey, Charles Yeo, “Cancer of the Pancreas,”. Principles & Practice of Oncology, 11th ed. Publication pending.

2 Anatomy of the Pancreas, image, Wikipedia, https://en.wikipedia.org/wiki/Pancreas.

3 Pancreatic Cancer, image, Go4Insurance Biz, http://www.go4insurance.biz/overview-pancreatic-cancer/.

4 Winter, Brody, et al, “Cancer of the Pancreas”.

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of a tumor.5 A CAT scan is usually performed to screen for pancreatic abnormalities suggestive of tumor growth. Even so, most pancreatic cancer can only be confirmed through post-surgical pathology. At that point, it is likely that the cancer cells have spread, increasing the chances that the cancer will metastasize and/or recur.6 Therefore, curing pancreatic cancer through the only known treatment is very rare as it is diagnosed at a point when it may no longer be localized. While chemotherapeutics such as 5FU have been shown to extend patient lifespan by 2 years, the cancer may eventually grow resistant to the treatment, leading to patient death. Radiation therapy is also a viable treatment option for localized tumors, though it is still not a cure for the deadly disease once it has metastasized.7 In short, while there are many treatments to extend patient lifespan through temporary fixes for growing tumors, there is no cure for pancreatic cancer.

REACTIVE OXYGEN SPECIES

Pancreatic cancer cells typically develop under the hallmark conditions of low-nutrient availability, which leads to metabolic stress in tumor cells. As a result, there is an increase in the production of reactive oxygen species (ROS), or free-floating oxygen molecules, also known as free radicals, in cells. ROS levels increase in low-nutrient, or more specifically, low-glucose environments, due to an incomplete reduction of oxygen in the mitochondrial electron transport chain during cellular respiration.8 Increased ROS levels result in an increase in oxidative stress levels within cells, thereby damaging cell repair mechanisms, leading to mutations in DNA, as seen in Figure 3, and eventually cell death.9 Therefore, they are a naturally occuring threat to cancer cell viability. Using this mechanism, chemotherapy treatments treat cancer by artificially causing an increase in NADPH oxidase, which also leads to an increase in ROS levels.10 This leads to DNA damage, causing tumor cell death. Therefore, ROS is an important mechanism in combating cancer cell viability and proliferation.11

HUR TRANSLOCATION

Cancer cells combat the presence of excess ROS, and therefore remain viable by upregulating enzymes which are dependent on nicotinamide adenine dinucleotide phosphate (or NADPH).

HuR (ELAVL1) is an RNA binding protein

Figure 3

Ibid.

Ibid.

7 Ibid.

8 Francesco Ciccarese and Vincenzo Ciminale, “Escaping Death: Mitochondrial Redox Homeostasis in Cancer Cells,”. Frontiers in Oncology. June 9, 2017.

9 DNA damage, image, http://www.emdmillipore.com/US/en/life-science-research/ antibodies-assays/assays-overview/apoptosis/dna-damage-tunel-assays/2_2b. qB.TgEAAAFA2jo.1ZdR,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F. 10 Ciccarese and Ciminale, “Escaping Death: Mitochondrial Redox Homeostasis in Cancer Cells,”.

11 Ibid.

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and its synthesis and translocation is an acute stress response.12 PDAC cells enhance cellular antioxidant defense when under metabolic stress caused by hypoglycemia by HuR translocation.

Once it has translocated to the cytoplasm, HuR binds to the antioxidant enzyme, isocitrate dehydrogenase-1, or IDH1, as shown in Figure 4.13 Thus, HuRmediated IDH1 downregulates excess Figure 4 ROS in stressed cells, thereby allowing the cells to survive.14 Therefore, the downregulation of IDH1 should decrease PDAC cell viability.

IDH1 downregulation could be tested with the IDH1 inhibitor FSM-206. Thus, if IDH1 inhibition decreases PDAC cell viability and FSM-206 is an IDH1 inhibitor, then cell viability should decrease as FSM-206 concentration increases.

METHODS AND RESULTS

MiaPaCa2 cells were cultured and plated in double triplicate in two 96-well plates at 1000 cells per well. Passage 16 was used for this experiment. Cells were incubated at 37°C and 5% CO2 in standard DMEM (containing 25 mmol/L glucose) under standard culture conditions, to allow cells to adhere to wells and stabilize. Cells were then incubated for 24 hours in low-glucose DMEM (containing 5 mmol/L glucose) to simulate low-nutrient pancreatic adenocarcinoma tumor microenvironment. Cells in one plate were treated with varying concentrations of FSM-206 (0, 1, 10, 50, 100, 1000, 2000, 5000, and 10000 nm). Cells in another plate were treated with varying concentrations of 5FU (0, 1, 10, 50, 100, 1000, 2000, 5000, and 10000 nm). Varying drug concentrations for FSM-206 and 5FU were created by mixing varied drug amounts with low-glucose DMEM. Treatments were refreshed daily to avoid excessive toxicity due to lownutrient environment. Plates were tested by PicoGreen Analysis to Figure 5

Ibid.

13 Anjali Sukhavasi, HuR Translocation to Cytoplasm to Bind with IDH1, image.

14 Mahsa Zarei, Shruti Lal, Seth J. Parker, Avinoam Nevler, Ali Vaziri-Gohar, Katerina Dukleska, Nicole C. Mambelli-Lisboa, Cynthia Moffat, Fernando F. Blanco, Saswati N. Chand, Masaya Jimbo, Joseph A. Cozzitorto, Wei Jiang, Charles J. Yeo, Eric R. Londin, Erin L. Seifert, Christian M. Metallo, Jonathan R. Brody, and Jordan M. Winter, “Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells,” Cancer Research. August 15, 2017: 2, PubMed. 12

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observe cell viability. Results are shown in Figure 5.

DISCUSSION

The results were inconclusive. The inconsistent correlation between the concentration of the clinically successful drug 5FU and the PDAC cell viability suggests large errors in plating the MiaPaCa2 cells. Therefore, the cell viability data obtained for the cells treated with FSM-206 is inconclusive.

This experiment must be repeated with more consistent plating, culturing and drug treatment technique to avoid contamination and variation in cell viability in plates. In addition, a DCF assay should be conducted to observe the fluctuation of ROS levels within cells treated with FSM-206 to test whether the cell viability decreases as a result of increased ROS levels. Finally, a rescue assay should be conducted using alpha-ketoglutarate to test whether FSM-206 is indeed an IDH1 inhibitor. Again, all of these assays should be conducted with extreme attention to pipetting and plating to ensure lack of variation in cell viability.

The mechanism that allows PDAC cells to survive under conditions of hypoglycemia also causes them to be resistant to chemotherapy treatment. As the upregulation of IDH1 downregulates ROS in cells, it interferes with the functional mechanism of chemotherapy, which causes cell death through increased ROS levels. Thus, the downregulation of IDH1 should lead to better chemotherapy performance.15 IDH1 can be downregulated by the drug FSM-206, an IDH1 inhibitor. The drug is tested in tandem with the chemotherapy combination drug 5FU with a control of the 5FU without the IDH1 inhibitor drug. If IDH1 downregulation ameliorates chemotherapy performance by reducing antioxidant defense, then the IDH1 inhibitor drug will cause 5FU to effect greater DNA damage that will result in PDAC cell death. To this end, FSM206 may not be used independently, but in conjunction with chemotherapeutics such as 5FU to ameliorate the effects of chemotherapeutic treatment. Further synergy testing could be conducted to observe potential synergy between chemotherapeutics and FSM-206.

15

Zarei, Lal, et al. “Posttranscriptional Upregulation of IDH1,” 2-3.

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TAYLOR TRAPP ’19 Taylor Trapp is a senior at Baldwin who lives in Philadelphia, PA. She has been attending Baldwin since the 1st grade and is the co-head of Florilège, representative of the Athletic Association, participant of Black Student Union and is a School Ambassador in Lamplighters. Taylor is also a member of the Varsity Volleyball and Swim teams. In her spare time, Taylor enjoys playing club volleyball, researching in labs and participating in multiple church activities such as the News Team and Dance Team.

Role of Mitochondrial Haplogroup Variants Determine Mitochondrial Dysfunction and Clinical Outcome in Pediatric Sepsis Taylor Trapp ’19 Dr. Francis McGowan John McCann Dr. Scott Weiss Children’s Hospital of Philadelphia Department of Anesthesiology and Critical Care Medicine STEMPREP 2018

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BACKGROUND

In 1965 the first polynucleotide, tRNA was sequenced although it was not identified.1 It took 7 years to isolate 1 gram of the tRNA from Baker’s yeast. Then they were sequenced through fragmenting into short oligonucleotides by various RNase enzymes for reconstruction and identifying by two dimensional chromatography and spectrophotometric procedures.2 At that time investigators were only able to identify a few base pairs per year which was not nearly enough to sequence a whole gene. Allan Maxam and Walter Gilbert then used chemical degradation on DNA, chemically cleaving specific bases of terminally labeled fragments which were separated by electrophoresis.3 This led to the method used by Sanger in which primer extension and chain termination methods were used for sequencing polynucleotides longer than oligonucleotide length.4 The Sanger method became more prevalent because of the elimination of the use of radioactive isotopes and in favor of fluorescent dyes which were much safer and more convenient. The first generation of automated sequencers used the Sanger method for single reaction sequences rather than the original four step protocol. With the development of the PCR method, using thermal cyclers, and enzymes such as Taq polymerase, (from Thermus Aquaticus) it was possible to sequence particular regions of interest.5 New sequencing methods continued to emerge and these new methods were referred to as Next Generation Sequencing (NGS), and were designed to produce massive amounts of sequence from multiple samples.

Next Generation Sequencing allows researchers to zoom into certain points and targets.

It also opens the regions for examining genomic or mitochondrial DNA, cDNA or RNA to compare differences in subjects on several different disease processes. The purpose of NGS in this case is to attempt to replicate previous results that were found by other scientists. This assay will determine if the results obtained prior to my work are accurate and conclusive with the ability to move forward to perform further assays on different samples. This experiment will allow testing of plasma samples from subjects with and without sepsis. For this study we used a sample from a known subject that has been tested several times as an internal control. We have been blinded from knowledge of which samples were normal versus control. By being blinded it made us have an unbiased opinion when carrying out the assay.

We used this technique in these experiments to specifically monitor sepsis in patient samples and to identify the different bacterium that initiated the sepsis. Sepsis is a life threatening complication of infection. It is seen with three different stages; sepsis, septic shock, and severe sepsis.

This fatal infection occurs when bacteria are released into the bloodstream, known as septicemia.

Although sepsis is commonly caused by an infection, it is in fact a condition of its own. This bacterial infection is very often misinterpreted or misdiagnosed with a normal infection that can be treated and restricted to its original spot origin. The difference between a normal infection and sepsis is that Holley RW, Apgar J, Everett GA, et al: Structure of a ribonucleic acid. Science. 1965;147:1462–5.)

RajBhandary UL, Kohrer C: Early days of tRNA research: Discovery, function, purification and sequence analysis. J BioSci. 2006;31:439–51.

3 Maxam AM, Gilbert W: A new method for sequencing DNA. Proc Natl Acad Sci USA. 1977;74(2):560–4. PMID: 265521 4 Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463–7 5 Saiki RK, Scharf S, Faloona F, et al: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science.1985;230:1350–4.

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in sepsis the infection enters the blood and spreads through the body and affects at least two organ systems. Sepsis can start with almost any type of infection, ranging from minor infections (athlete’s foot, urinary infection, abscessed tooth) to major infections such as meningitis. Septicemia is what leads to sepsis, which is why they are commonly used with one another.

Sepsis is very common in people of various ages, but most commonly found in very old or young people and people with certain risk factors. These risk factors include a weakened immune system, diabetes, kidney or liver disease, and AIDS. It is known that any infection may lead to sepsis such as: abdominal infection, pneumonia, or skin infection. In newborns, sepsis is called neonatal sepsis, and occurs within a month of birth. Treatments for neonatal sepsis depend on when the child developed it, whether early onset or late onset. Premature babies are more likely to develop sepsis due to their immune system not being completely mature. This is the same with babies who are not premature, but are low weight. Although symptoms in babies may be hard to determine, some signs may include low body temperature, apnea, fever, and a development of pale color.6 On the other hand, sepsis in seniors usually develops because as people age, the immune system seems to weaken. In a study shown in 2006, 70% of the people who had sepsis were patients over the age of 65.7 In addition to age, sepsis also occurs in seniors due to illnesses such as diabetes, kidney disease, cancer, and high blood pressure.

Some symptoms include a fever which is usually above 101 degrees or a temperature which is usually below 96.8 degrees.8 Another symptom of sepsis is rapid heart beat which ranges from 90-91 beats per minute, unlike the typical average of 70. An increase in the breathing rate is also seen to occur when looking at patients who may exhibit sepsis. In severe cases of sepsis, there may be symptoms such as discolored skin on certain parts of the body, a change in the mental capability, and a decrease in being able to use the bathroom.9 Along with these symptoms also comes extreme weakness and abnormal heart functions.

If sepsis progresses or isn’t treated, it can lead to septic shock which causes a substantial decrease in blood pressure, increased heart rates, altered mental status, and possibly the need of a ventilator.10 As time has progressed, the number of people with sepsis has increased in the United States. It was proven that sepsis causes more deaths in the United States than prostate cancer, breast cancer, and AIDS combined with one another.11 The reason for the increase in death is an aging population.

The mitochondria is an intracellular organelle that is known to be the powerhouse of the cell.

Its main job is to produce ATP through the process of cellular respiration. Mitochondria also regulates cell death, gene expression, and numerous cell signaling enzymes. Mitochondrial functions are abnormal in sepsis and contribute to injury and death.

Sepsis: Symptoms, Effects, and Causes. (n.d.). Retrieved from https://www.healthline.com/health/ sepsis#newborns 7 Ibid.

8 Sepsis. (2018, January 03). Retrieved from https://www.mayoclinic.org/diseases-conditions/sepsis/symptomscauses/syc-20351214 9 Sepsis: Symptoms, Effects, and Causes. (n.d.). Retrieved from https://www.healthline.com/health/ sepsis#newborns 10 Sepsis. (2018, January 03). Retrieved from https://www.mayoclinic.org/diseases-conditions/sepsis/symptomscauses/syc-20351214 11 Ibid.

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Mitochondria have their own genome which is maternally inherited.

Mitochondrial DNA is circular and much smaller than nuclear DNA. MtDNA codes primarily for some of the mitochondrial enzymes that produce ATP. Mitochondrial DNA haplogroups are a result of single mutations in the mtDNA sequence that have been passed maternally from mother to her child. In addition, mitochondria from different mtDNA haplogroups have differences in (Figure 1) mitochondrial function, substrate use, ATP Figure 1 displays the mitochondria and its components. generation, free radical production, and regulation of gene expression. This connects to sepsis because the mitochondria was tested on in this test.

RESEARCH DESIGN:

The first steps in our assay involve the combining of an unknown sample at a volume

equivalent to 70 nanograms of DNA, 5 microliters ION Shear 10x Reaction Buffer (from ION Torrent kit), and 35 microliters Nuclease Free Water, with minus the volume of unknown sample.

To the samples 10 microliters of Ion Shear Plus Enzyme Mix II were added for total volume of 50 microliters. The DNA samples were mixed by gently pipetting up and down using a microliter pipette. The samples were incubated at 37 degrees celcius for 9 minutes using thermal cycler. Five microliters of Ion-Shear Stop Buffer were immediately added and mixed by vortexing for at least 5 seconds followed by pulse spinning. Lastly, the reaction was stored on ice. To each sample was added 99 microliters of Agencourt AMPure XP Reagent magnetic beads. This mixture was pipetted up and down 5 times to mix the bead suspension then pulse-spun. The mixture was incubated at room temperature for 5 min. After incubation the tubes were pulse-spun and placed on magnetic rack for 3 minutes or until the solution was clear. After the solution cleared, the supernatant was removed carefully using a microliter pipette and discarded. This had to be done carefully in order to avoid disturbing the beads. Samples were washed by the addition of 500ul of 70% ethanol to each tube for 30 sec, turning the tube twice while on the magnetic rack. Once the solution clears, remove and discard the supernatant without disturbing the pellets. The washing step was repeated with ethanol and turning the tubes twice. Any residual ethanol was removed by pulse spinning, placing back on the magnetic rack and drawing it up with a microliter pipette. The magnetic beads were then air-dried for 5 minutes at room temperature. The tubes were removed from the magnet and 25 microliters of Low TE were added to the beads to elute the DNA from them and were mixed up and down with a pipette gently. They were then pulse-spun and placed on the magnetic rack for 1 minute or until the solution cleared. The supernatant that contained eluted DNA was transferred into 0.2ml PCR tubes. The samples were then barcoded using known

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Role of Mitochondrial Haplogroup Variants Determine Mitochondrial Dysfunction and Clinical Outcome in Pediatric Sepsis | Taylor Trapp â&#x20AC;&#x2122;19

barcoded adapters. This was done by adding 25 microliters of DNA, 10 microliters of 10x Ligase Buffer, 2 microliters of Ion P1 Adapter, 2 microliters of Ion Xpress Barcode X, 2 microliters of dNTP mix , 49 microliters of Nuclease Free H2O, 2 microliters of DNA Ligase, 8 microliters of Nick-Repair Polymerase which makes a total of 100 microliters. They were then placed in the thermocycler at 25 degrees for 15 minutes, 72 degrees for 5 minutes, and 4 degrees for up to an hour. The reaction was then transferred to 1.5ml LoBind conical tube and mixed by pipetting up and down, then pulse-spinning. They were incubated for 5 min at room temperature. 100 microliters of magnetic beads were placed into the sample and put on the magnetic rack for 5 minutes after being pulse spun. The supernatant was removed and discarded as waste. 500 microliters of ethanol were added to the sample and incubated for 30 seconds while turning the solution on the magnetic rack. When the solution cleared, the supernatant was removed and the following two steps were repeated. The ethanol was removed, pulse-spun, and placed on the magnetic rack to air dry for 5 minutes. The tubes were removed from the magnetic rack and 20 microliters of Low TE buffer to the beads and pipetted up and down to mix. They were then placed on the magnetic rack for 1 minute before removing the supernatant to 1.5ml LoBind conical tube. The last steps to the research design included analyzing the Purity of DNA using the Agilent 2100. The DNA High Sensitivity dye concentrate (blue cap tube) and High Sensitivity gel matrix (red top tube) sat at room temperature to equilibrate for 30 minutes. 15 microliters of High Sensitivity were added to the High Sensitivity gel matrix and vortexed to mix. They were then pulse spun down before transferring them to a spin filter and centrifuging for 10 minutes. The dye then had to sit at room temperature to equilibrate. A new High Sensitivity chip was placed on the chip priming station and 9 microliters of gel dye mix was placed into the well with the dark G. It was important to make sure the syringe on the priming station was at 1 ml. The next step to this experiment was closing the priming station lid in order to plus plunger down for 1 minute. This allows the solution to spread throughout the chip. The chip was released and pulled back up to 1 ml after waiting for 5 seconds. The chip priming station was then opened to pipette 9 microliters of gel-dye mix into the wells marked with the light G. 5 microliters of the marker was placed in all dample and ladder wells which was 1-11. Next, 1 microliter of ladder was placed into the well marked 12. In wells 1-3, samples SW, 1, and 4 were placed. In the wells 4-7, sample 4, 6, and 7 were placed. In the wells that do not contain sample, 1 microliter of marker was placed. The chip was then ran on the Agilent 2100 Bioanalyzer within 5 minutes using the dsDNA 7500 program.

THE BALDWIN REVIEW 2018

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Role of Mitochondrial Haplogroup Variants Determine Mitochondrial Dysfunction and Clinical Outcome in Pediatric Sepsis | Taylor Trapp â&#x20AC;&#x2122;19

RESULTS:

In the first experiment, the base pairs appeared around 70. After a series of experiments to raise the number of base pairs, they finally appeared around 300, which was right in the middle of where we expected.

Over a series of experiments, we found that optimal library amplification from small volume, human blood mtDNA samples required: -

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Reducing fragmentation incubation time from 9 min to 5 min Using approximately 50 nanograms of input DNA And PCR primer sets designed and optimized to generate larger amplicons of 300 base pairs

(Figure 2) Figure 2 shows a gel electrophoresis of the amplified DNA fragments from different patient samples. (Lanes 4, 7-10) or degraded and/or incorrect products (2, 3, 5, 6)

Using this combination of factors we were able to consistently contain products with a uniform size of approximately 300 base pairs and no degradation.

The results came out as we wanted during the second time of the experiment. By completing the steps previously mentioned, the gel was able to reach 300 base pairs. This means that the test came out to be correct.

(Figure 3) Figure 3 displays the gel electrophoresis after using the combination of factors to improve them to 300 base pairs.

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