THE SHOOTING METHOD: AN EFFECTIVE TOOL FOR BOUNDARY VALUE PROBLEMS IN ODE

The shooting method is a numerical approach used to solve boundary value problems (BVPs) for ordinary differential equations (ODEs). It works by converting a BVP into an initial value problem (IVP), allowing the use of established IVP solvers like the Euler or Runge-Kutta method to iteratively find solutions. The process will begin with an initial guess for the derivative at one boundary, followed by solving the IVP and adjusting the guess based on how the computed solution aligns with the desired boundary condition at the other end. This research project discussed the theoretical foundations, practical implementation, and convergence characteristics of the shooting method, emphasizing its advantages and limitations relative to other numerical techniques. Through illustrative examples using MATLAB programing scripts, we showcased the method\u27s effectiveness in tackling complex BVPs, highlighting its importance in computational mathematics

APPLICATION OF CRISPR TO REMOVE MULTIPLE BETA-LACTAMASE GENE VARIANTS**

In modern healthcare settings, antibiotic resistant bacteria are increasingly becoming an issue. Bacteria are becoming resistant at a faster rate than we can develop drugs for, which has lead to alternatives being sought out. A solution could be to remove resistance using gene editing technology like CRISPR. This study focused on β-lactamases specifically, which confer resistance to some of the most common drugs. These enzymes work by hydrolyzing the β-lactam ring in penicillin and cephalosporin class antibiotics, and are divided into four classes based on protein sequence. It can be assumed that the active site of β-lactamases within each class are similar, supported by the fact that 3 of the 4 classes all use a serine amino acid as the core component of the active site. By applying CRISPR-Cas9, this paper examines the potential to remove multiple gene variants using a single guide RNA. The plasmids pHflu2 and pETcon-NK were used, which contain the genes for β-lactamases TEM-116 and TEM-1 and chloramphenicol and kanamycin resistance, respectively. TEM-116 and TEM-1 are variants of the TEM gene, which is a class A β-lactamase. These plasmids were inserted via electroporation into the Escherichia coli strain LC-EO3, which contains a Cas9 gene, to generate two cultures of LC-EO3 each containing one of the β-lactamase plasmids. A plasmid containing a guide RNA that targets TEM-116 was synthesized by Sigma Aldrich and called pAmpR (SRID). Plasmid pAmpR (SRID) also contains an ampicillin resistance cassette. The LC-EO3 strains containing pHflu2 and pETcon-NK will be transformed by electroporation with pAmpR (SRID). These strains will then be replica plated on Luria agar containing ampicillin, chloramphenicol or kanamycin. It is expected that the Cas9, guided by one sgRNA will cause double strand breaks in pHflu2 and potentially pETcon-NK, causing the bacteria to become sensitive to the antibiotics chloramphenicol and kanamycin

EVALUATION OF MYOGLOBIN LEVELS IN THE LOCOMOTER MUSCLES OF FLORIDA MANATEES

Diving marine mammals typically have higher levels of myoglobin protein within their locomoter muscles to increase storage of oxygen during breathhold episodes. The oxygen that is stored is used for ATP production to power swimming activity. With limited information on the levels of myoglobin in the swim muscles of Florida manatees, this research focuses on determining the quantity of myoglobin in collected locomoter muscle from stranded manatees of different age groups. Collected results reveal that the Florida manatee have lower swim muscle myoglobin levels than the more robust swimming juvenile bottlenose dolphin (4.74 mg/g and 25.84 mg/g, respectively). Furthermore, the myoglobin levels of Florida manetee calves are significantly lower than adult animals (0.603 mg/g) suggesting these animals are immature at birth and the concentration increases during development. Given that manatees are more slow-moving herbivores and myoglobin plays a role in energy storage, lower muscle myoglobin levels in manatees compared to dolphins could be a reflection of their less active lifestyle. However, manatees could also rely on other ATP generating pathways within the skeletal muscle to additionally fuel diving episodes. Future research will focus on further characterization of the development of myoglobin levels in other age groups of the florida manatee and also evaluate the roles of additional energy producing pathways (e.g. anaerobic respiration) used by the locomoter muscles to power swim activity

ALLOMETRIC GROWTH IN THE EASTERN BOX TURTLE, TERRAPENE CAROLINA CAROLINA

Allometry examines how changes in body parameters are correlated with changes in overall size. There are few studies that have been conducted specifically on the allometric relationships of shell measurements of adult Eastern Box Turtles (Terrapene carolina carolina). Of these studies, only time allometry is examined focuses on growth studies of T. c. carolina. Allometric growth could understand the different growing states of the turtle. Many growth studies focused on the factors affecting growth of the whole body of the turtle, and they do measurements on carapace length and width and compared it to other factors. The study site that was used in this study was Tumbling Creek Woods in Gainesville, GA, and this study started in May 2013 and is ongoing. The study design is a hybrid design with radio telemetry and monitoring with capture mark and recapture. We used calipers to measure carapace length, carapace width at 2/3 scutes or max, plastron length, and height of the turtle. We compared measurements of males and females to see if there was a relation within between the measurements. Data analysis suggested some relation between carapace length and width, as well as sexual dimorphism

CHARACTERIZATION OF THE INTERACTION BETWEEN ADENOVIRAL PROTEIN E4 11K AND THE DNA-PK-DEPENDENT DNA DAMAGE RESPONSE**

Adenovirus (Ad) can cause upper respiratory infections, gastroenteritis, and conjunctivitis. The linear, double-stranded DNA Ad viral genome can be broken up into early and late phase genes. Our protein of interest, E4 11k, is a product of one of the early genes that works to disrupt normal cellular function to enhance the viral replication cycle. One of the mechanisms E4 11k uses to achieve this disruption is through the inhibition of the DNA damage response (DDR). The DDR is responsible for repairing double-stranded DNA breaks (DSBs) through non-homologous end joining (NHEJ). During DDR, DNA-dependent protein kinase (DNA-PK) becomes activated via phosphorylation. During an infection, Ad’s genome triggers the DSB repair pathway due to the genome’s structural similarity to a cellular DSB. The DDR tries to “repair” the viral genome by ligating the viral genomes into concatemers, disrupting viral DNA replication. Ad, however, has evolved mechanisms to prevent activation of the DDR. Previously, E4 proteins have been shown to regulate the activation of DNA-PK. In this study, we aim to determine E4 11k’s ability to inhibit phosphorylation of DNA-PK in response to cellular DSBs. HeLa (cervical cancer) cells were treated chemically with different combinations of either etoposide (drug that induces DSBs), Nu771 (inhibitor of DNA-PK phosphorylation), or an Ad E4 11k only viral infection. The phosphorylation status of DNA-PK will be analyzed through immunoblotting. In the future, we want to characterize DNA-PK localization during an Ad infection. Immunofluorescence microscopy will be used to visualize protein localization. Our initial findings indicate a 97% reduction in DNA-PK activation. It has previously been shown that etoposide treatment combined with the inhibition of DNA-PK activation result in cancer cells that have less adherence. These findings suggest a potential mechanism for which E4 11k can be used as a novel chemosensitizer for therapy-resistant cancer cells

FINDING EQUILIBRIUM CONSTANT VALUES FOR THE ETHANOL-ACETIC ACID REACTION

The ethanol-acetic acid reaction is often used in first-year chemistry classes to illustrate the importance of a catalyst and what occurs at chemical equilibrium. The impact of adding different catalysts and the time required for the reaction mixtures to reach equilibrium were studied. In all cases, 0.30 moles of ethanol and 0.30 moles of acetic acid were used. It is concluded that students may learn several things from this reaction. Four of these are: 1) the importance of adding a catalyst, 2) how the concentration of reactants decrease and products increase with time, 3) how to use titrations to measure the concentration of the remaining acetic acid and to use this to determine the concentration of the ethanol and ester concentrations and 4) how to measure an equilibrium constant. This reaction has the added benefit of allowing students to use titrations to track the decrease in reactant concentration with time. In one trial, initial product (ester) molar concentrations of 6.2, 6.9, and 6.9 were computed for one, two, and three weeks after the catalyst was added, yielding an equilibrium constant of 2.0

LIKELY PATHOGENICITY OF D119A AND K146N VARIANTS OF HUMAN IDUA

Mucopolysaccharidosis type 1 (MPS1) is a disorder of abnormal accumulation of toxic glycosaminoglycans (GAGs) in various tissues. It occurs due to a decrease in the activity of alpha-L-iduronidase (IDUA), which hydrolyzes terminal GAG alpha-L-iduronic acid residues. D119A and K146N are existing human IDUA variants of unknown significance with respect to MPS1 development. Both variants are in critical-domain regions of existing protein models and involve substitutions that change chemical class. To assess the potential clinical significance of these variants we used in silico evolutionary conservation, multiple sequence alignment, protein structure, and molecular dynamic simulation (MDS) tools. Aligning 29 different animal IDUA sequences revealed high conservation of aspartic acid 119 and lysine 146 compared to surrounding regions, suggesting purifying selection on these amino acid positions. We then used predictive software that analyzed the potential deleterious effect of variants D119A and K146N. These tools also suggested a low tolerance to substitutional change for the two variants and their likely pathogenicity. Root-mean-square-deviation (RMSD) MDS analysis revealed increased protein flexibility in D119A and K146N (1.343 & 1.464, respectively) over time compared to wild-type (1.276). Root-mean-square-fluctuation (RMSF) MDS analysis showed widespread conformational changes in both variants, with some differences in regions critical to enzyme function (e.g., alpha-L-iduronate and beta-D-mannose binding). Overall, our results support that both D119A and K146N variants are likely damaging and might explain the presence of symptoms consistent with MPS1 in some patients that don’t meet present genetic criteria. In vivo experimentation in a model organism should now follow to confirm the impacts of these variants on MPS1 and GAG homeostasis

Field observations of gopher tortoise (Gopherus polyphemus) activity at a rare double-burrow

After a review of the journal, it does not appear that abstracts are included with Research Notes. Therefore, no abstract is provided. However, we certainly defer to the Editors on this requirement and will happily provide an abstract if needed

DESIGN OF A HIGH VACUUM SYSTEM USING SOLIDWORKS

For our rotational spectroscopy research, a high vacuum system is designed to accomodate the large Fabry-Perot cavity resonator for a Balle-Flygare type Fourier transform micowave (BF-FTMW) spectrometer. To cover a broad frequency span of 3 - 40 GHz, the diameter of MW reflection mirrors are ~20 inches. A large vacuum chamber with a torispherical cap on the one end is designed using the Solidworks. Mechanical components with various industrial standards, including ASME, ISO, DIN, and JIS are used in this vacuum system degsign to compatible with all sorts of research and industrial standards

THE EFFECT OF CANNABINOIDS ON THE DEVELOPMENTAL MORPHOLOGY OF ZEBRAFISH: A SUMMARY**

Results from the past few years on exposure of zebrafish embryos to varying concentrations of delta nine-tetrahydrocannabinol (Δ9-THC), Δ8-THC and cannabidiol demonstrated delayed hatch rate, higher death rate, lowered heart rates and shorter embryos with curved spines when compared to control embryos. We also observed alterations on morphology of jaw structures though have yet to see a consistent pattern. In this study we are processing embryos from last year stored in 100% methanol and are using Alcian Blue staining to examine jaw structure in an effort to identify a consistent pattern of abnormality. We will also analyze data across all three years to summarize overarching trends