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Poster Abstracts

 

2020 Poster Abstracts

Agricultural Biotechnology

Evaluation of Contact Independent Inhibition of Fungal Growth on Lemons and Tomatoes by Rhodococcus rhodochrous DAP 96253


Dr. George E. Pierce and Muzna Saqib Georgia State University BACKGROUND Fungal fruit rot plays a major role in postharvest fruit processing. Under a typical fungicide program, fungal infection has been shown to significantly contribute to fruit rot, accounting for losses of up to 15% of product yield. Additionally, common fungicidal chemicals display various levels of toxicity and may leave residues on fruit, making them undesirable for consumption. Fruit is typically stored at cold temperatures to prevent rot and delay ripening. However, cold storage often results in chilling injury which damages fruit and predisposes them to fungal infection. This is particularly common in citrus fruits which may be stored for several months or shipped over long distances. Investigation into alternative methods of controlling postharvest rot would have a significant impact on increasing yield and quality of fruit harvests. Rhodococcus rhodochrous is a non-pathogenic bacterium found ubiquitously in terrestrial and aquatic environments. Rhodococcus rhodochrous strain DAP 96253 has previously demonstrated the ability to reduce fungal spore germination. This report characterizes the inhibition of two common postharvest fruit rot pathogens Aspergillus niger and Fusarium solani on lemons and tomatoes via a contact independent mechanism using R. rhodochrous DAP 96253. METHODS A. niger and F. solani were grown on either potato dextrose agar or sabouraud dextrose agar. Spores were isolated in a buffer solution and used to inoculate lemons and tomatoes at various concentrations. Inoculated fruit were placed into a sealed headspace with various weights of R. rhodochrous DAP 96253 cell paste. Fungal growth was characterized over a two-week period. RESULTS Fruit placed within seal air space containing R. rhodochrous DAP 96253 cell paste had reduced levels of A. niger and F. solani growth. This antifungal effect tended to increase as cell paste weights increased. This antifungal effect is most prominent on tomatoes. Inclusion of activated carbon into this shared air space greatly diminished antifungal effects. CONCLUSIONS R. rhodochrous DAP 96253 fermentation cell paste displays contact independent antifungal properties on A. niger and F. solani grown on tomatoes and lemons. Inclusion of activated carbon into the shared air space of lemons or tomatoes and R. rhodochrous cell paste reduces antifungal effects. This indicates the involvement of volatile compounds in this antifungal process. Future experimentation will seek to identify these antifungal volatiles.




Genes critical for biofilm formation by Salmonella enterica serotype Tennessee


S Lee 1 and J Chen 11
Department of Food Science and Technology, The University of Georgia, Griffin, GA
BACKGROUND Biofilm formation is a strategy of Salmonella to survive in a hostile environment. Although it is well understood that biofilm offers Salmonella increased tolerance to stress and enhanced survival on low moisture food like seeds and nuts, the molecular mechanism underlying Salmonella biofilm formation and colonization on dry food have not been fully elucidated. The purpose of this study is to identify the genes that are involved in colonization and biofilm formation by Salmonella. METHODS Mini-Tn10 transposon mutagenesis was used in the present study to randomly interrupt the genes of Salmonella enterica serotype Tennessee, an isolate from the large, widespread peanut butter outbreak in 2007. The ability of selected Salmonella mutants in forming biofilms were compared with their wild type parent in a 24-well polystyrene tissue culture plate. Biofilm mass was quantified using the crystal violet binding assay. Mutants forming significantly less (P<0.05) biofilm mass in comparison to their wild type parent were selected. Genomic DNA of mutant cells were extracted and subjected to deep DNA sequencing. Specific gene in each mutant that was interrupted by mini-Tn10 insertion was identified by comparing the obtained DNA sequencing data with those deposited in the Genbank using BLAST search. RESULTS A total of 56 colonies of S. Tennessee mutants were obtained, and only 5 colonies were selected for further analysis according to the results of biofilm assay. Cells of the 5 mutants formed significantly lower (P<0.05) biofilm mass than the parent strain. Sequencing analysis revealed that the interrupted genes in collected mutants encode for bacterial cell membrane lipoprotein, DNA topoisomerase III, attachment invasive locus protein, bacteriocin immunity protein, or cell division protein. CONCLUSIONS The study identified some of the genes that play a role in the formation of biofilms by Salmonella. These genes could be likely targets for control of pathogen colonization on low moisture food.




Utilization of Imaging Flow Cytometry to Assess Extracellular Vesicle Internalization


BJ Jurgielewicz (1-3), Y. Yao (1,2), SL Stice (1-4).
(1) Regenerative Bioscience Center, University of Georgia(2) Department of Animal and Dairy Science, University of Georgia(3) Biomedical Health Science Institute, University of Georgia (4) Aruna Biomedical
BACKGROUND Extracellular vesicles (EVs) are nanosized lipid bilayer-bound vesicles, ranging between 50-1000nm in diamter, that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. EVs have the ability to modulate recipient cells through the delivery of genetic material. Despite the therapeutic potential of EVs, there is limited information on EV uptake kinetics and specificity. METHODS We optimized an imaging flow cytometry (IFC)-based platform to quantitatively assess dose, time, and recipient cell specificity effects on human embryonic kidney cell (HEK293T) EV internalization in a high-throughput manner. IFC provides both qualitative imaging and quantitative flow cytometry metrics to assess isolated EVs and EV internalization, in vitro. RESULTS HEK293T EV uptake is an active process that is dose and time dependent. Further, the selectivity of EV uptake was quantified in vitro, where HEK293T EVs were internalized at higher quantities by cells of the same origin. Lastly, neural stem cells internalized significantly more HEK293T EVs relative to mature neurons, suggesting that stem or progenitor cells, which are more metabolically active than terminally differentiated cells, may have higher rates of active EV internalization. CONCLUSIONS The characterization and high-throughput quantification of EV uptake including active internalization, specificity, dose and time dependence, and kinetic assays will help inform, develop, and optimize EV-based therapeutics.





Cell Therapy & Tissue Engineering

Extracellular Vesicles Mediate Neuroprotection and Functional Recovery after Traumatic Brain Injury


Min Kyoung Sun University of Georgia BACKGROUND The lack of effective therapies for severe traumatic brain injures (TBIs) leaves patients with lifelong disabilities. Therapeutic intervention in the acute phase of severe traumatic brain injury (sTBI) is needed to minimize secondary injury and improve prognosis. We hypothesize acute intravenous injections of neural stem cell derived extracellular vesicles (NSC EVs) following a sTBI can attenuate tissue damage, and exert neuroprotective effect via paracrine signaling and enhancing endogenous NSC repair activity. METHODS NSC EVs were collected, purified from human embryonic stem cell derived NSCs, and stored at -20°C until needed for treatments. Adult Sprague Dawley rats were subjected to controlled cortical impact (CCI) to induce severe left hind limb deficit. Subsequently, they received three intravenous injections at 6 hour, 24 hour and 48 hour-post injury according to their assigned groups: CCI only (n=16), vehicle-treated (n=16), or NSC EV-treated group (n=16). Functional recovery was measured via balance beam walk test at day 4, 7, 14, 21, and 28 post-CCI. At the end of 28 days post-CCI, immunohiostological assessments allowed to identify tissue level changes among groups. Linear discriminant analysis (LDA) was used to reflect overall recovery patterns in control and treatment cohorts. RESULTS We observed that male NSC EV-treated rats demonstrated significantly reduced lesion sizes, and enhanced presence of endogenous NSCs, as well as attenuated motor function impairments in comparison to CCI-control and vehicle-treated animals. Although statistically not significant, we also observed a therapeutic effect of NSC EVs on brain lesion volume, nestin expression, and behavioral recovery in female rats. CONCLUSIONS In conclusion, our study demonstrates the neuroprotective and functional benefits of NSC EVs for treating sTBI in male subjects but not in females. Further investigation is required to fully understand the sex-dependent molecular repair mechanisms of NSC EVs, effective dosage, and therapeutic administration windows. Considering our promising results, NSC EV-based treatment could provide an accessible and effective new treatment of sTBI patients in a clinical setting.




A Mesofluidic Bioreactor for Modeling Brain Organoid Growth and Differentiation


Seleipiri Charles Georgia Institute of Technology BACKGROUND Organoids are three-dimensional multicellular structures capable of recapitulating aspects of organ function. They produce anatomically relevant cellular structures and some functions that closely resemble those seen in vivo. As a result, they hold great potential as models for neuropsychiatric and neurodevelopmental diseases and personalized medicine applications. Despite the advances in generating organoids, the difficulty in reproducibly generating the same cell types and structures in these tissues hinders their use in screening applications. Additionally, the heterogeneity across the samples complicates conducting hypothesis-based screens, large-scale screens, and prevents observation of subtle phenotypes. Hence, there is a need to generate and culture these organoids in a more reproducible fashion to allow for reliable results during screening applications. Current methods of organoid culture are mostly limited by lack of in situ characterization. METHODS In this project, we address this issue using a microfluidic bioreactor. The bioreactor mold was designed and 3D printed using CAD software. The devices were then fabricated using poly-dimethyl siloxane (PDMS) via replica molding. The device was bonded to a glass slide to enable longitudinal imaging of the organoids and downstream analysis via fluorescence microscopy. Organoids were characterized via phase contrast imaging and immunohistochemistry (IHC) RESULTS We demonstrate the longitudinal capability of the bioreactor via functional imaging and image-based profiling. Longitudinally monitoring organoid differentiation and maturation provided information necessary for optimizing culture conditions, which was supported by results from end-point assays (IHC) CONCLUSIONS The microfluidic platform is ideal for this application because it allows for simultaneous and in situ monitoring of multiple samples making the process less tedious than using most conventional culture methods. We expect that this technology will help establish reproducible culture conditions for organoids thus enabling more robust screening.




A Synergistic Approach to Develop Antibacterial Electrospun Scaffolds Using Honey and S-nitroso-N-acetyl penicillamine


Sama Ghalei School of Chemical, Materials and Biomedical Engineering, University of Georgia BACKGROUND Bacterial infection has been increasingly recognized as the major reason for the failure of tissue engineering scaffolds. Therefore, there is a need for novel and multifunctional biomaterials that not only enhance tissue regeneration but also can combat infection. An antibacterial and bioactive scaffold was fabricated in this study by incorporation of honey and the nitric oxide (NO) donor, S-Nitroso-N-acetylpenicillamine (SNAP), into polylactic acid (PLA) nanofibers using a single jet electrospinning method. Honey (HN), the ancient healing medicine, is a natural antibacterial agent with high biocompatibility. NO is also known to be a potent antimicrobial agent and a critical player in the physiological processes such as vasodilation, angiogenesis, inflammation, tissue fibrosis, or immune responses. METHODS In this study, Polylactic acid/honey/SNAP (PLA/HN/SNAP) composite nanofibers were fabricated using electrospinning to create an antibacterial and bioactive scaffold. The morphology, mechanical properties, wettability properties, and in vitro NO release kinetics of the fabricated scaffolds were evaluated. In addition, antibacterial efficacy and the cellular activities of the 3T3 fibroblast cells on the nanofibrous scaffolds were investigated. RESULTS PLA/honey/SNAP (PLA/HN/SNAP) nanofibers had an average diameter of 624.92 ± 137.69 nm and showed a sustained release of NO for 48 h. The nanofibrous morphology, moderate wettability, and desirable tensile properties of PLA/HN/SNAP scaffolds proved that they can closely mimic the natural ECM and could be suitable for soft tissue engineering, such as skin and cartilage. The results of antibacterial studies revealed that the synergistic combination of honey and SNAP significantly reduced the viability of Staphylococcus aureus. In addition, qualitative and quantitative 3T3 fibroblast cell culturing experiments proved that the PLA/HN/SNAP scaffolds supported better cell attachment and proliferation compared to PLA. CONCLUSIONS This research provides preliminary work towards the development of a novel, multi-functional nanofibrous scaffold, and the results indicate that this material has great potentials for clinical tissue engineering applications, especially for the prevention of bacterial infections, skin wound healing, and damaged tissue regeneration.





 

Drug Discovery & Development

Additive Manufacturing to Create Antimicrobial Nitric Oxide Releasing Surfaces


Manjyot Kaur Chug University of Georgia BACKGROUND Each year 99,000 deaths are reported due to hospital-acquired infections. A great extent of nosocomial infections in the patients' source from medical devices that feature a suitable surface to promote biofilm formation. One approach to addressing these infections is to create bioactive devices with nitric oxide-releasing (NO) properties. Nitric oxide has been extensively utilized for treating medical device infections due to its inherent antimicrobial and anti-inflammatory properties. Polymers releasing NO at endogenous rates (ca. 0.5-4x10-10 mol cm-2 min-1) possess similar antimicrobial properties. To overcome the design of geometrically complex devices, additive manufacturing technology was utilized to print custom designed films (porous and solid) using polycarbonate-based silicone elastomer (ChronoSil). The films were impregnated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) using a solvent-swelling process to generate antibacterial interface. Based up on the modulation of structure via distinct porosities, the NO-releasing films were studied for real-time NO release, SNAP leaching and impregnation, and antibacterial properties, that are crucial to enable clinical translation. METHODS

Polycarbonate-based silicone elastomer (PCU-Sil) films were fabricated with different porosities (60% porous, 40% porous, solid, and capped) using 3D printer. Films were soaked in SNAP (100 mg/mL) dissolved in 40% methyl ethyl ketone and 60% methanol for 2 h and dried under ambient conditions for 24 h. The NO-releasing PCU-Sil were incubated in PBS-EDTA at 37°C to determine the real-time NO release under physiological conditions using Nitric Oxide Analyzer 280i (Frederick, CO). The SNAP impregnation and leaching studies for the 3D printed films were conducted using UV-Vis spectroscopy. SEM and EDS mapping were used to examine the morphology and elemental composition of the films before and after SNAP impregnation. Finally, NO releasing films were assessed for antibacterial ability using 24 h bacterial adhesion against Staphylococcus aureus strain of bacteria frequently found in medical device related infections.

RESULTS The UV-vis studies demonstrated highest amount of SNAP in porous samples (60% and 40%) followed by solid and capped. All the samples exhibited higher levels of NO release in the initial days. While the porous samples demonstrated continued NO release at relatively higher levels up to 14 days, solid and capped samples had diminished flux levels after day 5. NO releasing films exhibited > 99% suppression of S. aureus adhesion on the film surface (p < 0.05) after 24 h. CONCLUSIONS The SNAP impregnated 3D-printed PCU-Sil films provide > 7 d of NO release at physiologically relevant levels. This study highlights the importance of additive manufacturing in biotherapeutics and paves a new path for developing patient specific antimicrobial medical device with customized NO-releasing capability. The closely regulated personalized bioactive polymers can help overcome the design of geometrically complex devices, the potential toxicity to the patient from the biofilm colonization, and emergence of antimicrobial resistance.




Characterization of the Combined Therapy of a Nitric Oxide (NO) Donor Molecule and Cerium Oxide Nanoparticles for Antimicrobial Applications


Lori Estes

School of Chemical, Materials, and Biomedical Engineering, College of Engineering, University of Georgia

BACKGROUND Broad-spectrum antimicrobials are needed towards mitigating the issues of antibiotic-resistant infections. It is highly important to formulate new antimicrobials by combining agents with different mechanistic and broader microbial targets. A combined antimicrobial solution could be a highly critical step towards developing the strategy to prevent polymicrobial infection. Herein, we have investigated the interaction and antimicrobial potential of a solution that contains cerium oxide nanoparticles (CNP) and a nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP). It is hypothesized that these two agents induce synergistic effects and would provide broad antimicrobial effects since CNP is known to be an effective antifungal agent while NO released by SNAP is known to be a potent bactericidal agent. METHODS

Different concentrations of SNAP and CNP were combined in a solution and tested for colloidal stability, NO release, mammalian cell cytotoxicity, and antimicrobial efficacy against Staphylococcus aureus, Escherichia coli, and Candida albicans, accounting for Gram-positive, Gram-negative, and fungi, respectively.

RESULTS

The combination of SNAP and CNP led to decreased colloidal stability but increased biocompatibility compared to CNP and SNAP alone, respectively. The scavenging capabilities of CNP allowed for a prolonged NO release from the combination solution. SNAP and CNP combined in equimolar solution of 3 mM were found to be highly effective for all microbes tested compared to higher amounts of the treatments required individually.

CONCLUSIONS These results hold a promising outlook toward the development of broad-spectrum antimicrobial coatings and films with the potential to prevent polymicrobial infections and further enhance biomedical device usage and applications.




A Recipe for Academic Labs to Produce SARS-CoV-2 RT-qPCR Test Kits


Sara Fakhretaha-Aval

Georgia Institute of Technology

BACKGROUND The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 30 million people, claimed many lives, and substantially disrupted activities in the public and private sectors. Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. METHODS

Here we describe our in-house Master Mix and RT-qPCR assay for detection of SARS-CoV-2. We discuss preparation of singleplex and multiplex primers and probes with CDC sequences that can be used with commercial enzyme Master Mixes. In addition, we present the production of reverse transcriptase (RT), Taq DNA polymerase, and ribonuclease inhibitor (RI) proteins, and the formulation of a working 1-step enzyme Georgia Tech Master Mix (GT-Master Mix) for use with our primers and probes.

RESULTS

We formulated a functional SARSCoV-2 assay that compares favorably to commercially available RT-qPCR kits. Our assay comprises a Master Mix as well as primers and probes identical to validated CDC sequences. GT-Master Mix is composed of affinity-purified GT-rRI, GT-His-Taq, and GT-MMLV reverse transcriptase, a compatible buffer containing cationic cofactors, plus BSA and trehalose for stability and long-term storage. In GT- Master Mix, the efficiencies of our GT primer and probe sequences met (multiplex) or closely approached (singleplex) our efficiency target of 90- 110% with high linearity (r 2 >0.990), indicating minimal primer dimers or non-specific amplification. The efficiency and linearity of our multiplex kit over a five-log concentration range is competitive with other kits that have received EUA for SARS-CoV-2 testing. In sum, our GT RT-qPCR assay, composed of proteins and enzymes produced in house, with either singleplex or multiplexed primers and probes kit exhibits a high level of qPCR efficiency and storage stability.

CONCLUSIONS The goal of our project was to create contingency SARSCoV-2 diagnostic test components in the face of supply line insecurity. We translated published information about RT-qPCR and sophisticated commercial kits into a series of fundamental protocols, executable with consumables and equipment routinely used in academic biochemistry laboratories. Our published blueprint should be readily reproducible by research teams at other academic institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource limited settings. In the long term, our protocols should be adaptable to the detection of other novel or seasonal infectious viral agents.




Rapid Susceptibility Testing from Positive Blood Cultures


Alexandra Filbrun

Georgia Institute of Technology

BACKGROUND In large part due to the rapid development of antimicrobial resistance, bacterial bloodstream infections (BSIs) remain a major cause of mortality and morbidity despite the wide availability of antibiotics. With exceedingly low bacterial burdens of 1-100 colony-forming-units per mL blood, conventional diagnosis relies on lengthy blood culturing and purification steps prior to already slow identification and antibiotic susceptibility testing (AST). This >60-hour time-to-result imposed on actionable treatment determinations negatively impacts patient outcomes and increases proliferation of antimicrobial resistance through the misuse and overuse of broad-spectrum antibiotics. Consequently, the development of novel technologies capable of rapidly recovering bacteria originally present in high blood backgrounds is increasingly important. METHODS

To address this need, we developed a novel bacterial separation technology from positive blood cultures that couples selective lysis with direct, centrifugation-based bacterial recovery.

RESULTS

Evaluated against the most common BSI-causing bacteria pathogens: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria can be recovered in <15 minutes with minimal sample handling. ASTs are readily performed with excellent correspondence to much slower ASTs performed with VITEK-2 Instrumentation.

CONCLUSIONS Overall, this direct from positive blood culture AST enables susceptibility determinations to be assessed in 3-6 hours from blood culture positivity, with minimal sample handling and processing.




Functional Outcome and Self-reported Outcome Comparison Between Osseointegrated and Socket Prosthesis for Lower Extremity Amputees: A Meta Analysis


Jacob Lonowski

Georgia State University Department of Physical Therapy

BACKGROUND

Osseointegrated (OI) prostheses are becoming a more attractive option for patients with an amputation, especially for those who have had chronic skin issues and recurrent infections with socket prosthetic use. Despite the mounting number of publications which support OI prostheses, a meta-analysis of related outcomes has not yet been conducted. Thus, this study's aim is to provide a meta-analysis of self-reported and functional outcome comparisons between OI prostheses and traditional socket prostheses in lower extremity (LE) amputees. The selected functional outcomes for comparison, the 6 Minute Walk Test (6MWT) and Timed Up and Go (TUG), are staples for assessing the functionality of an individual and are valid and reliable measurements. The self-reported outcomes, the Questionnaire for Persons with a Transfemoral Amputation (QTFA) and the Short Form 36 (SF-36), are both valid and reliable measures for the assessment of this population. Both the QTFA and the SF-36 are questionnaires which assess the status of an individual across a spectrum of physical and mental qualities.

METHODS

Six databases were searched including Cochrane, Pubmed, Medline, EMBASE, CINAHL, and Web of Science. Literature search was independently conducted by two reviewers. 1434 studies met the search criteria, 277 studies screened after the removal of duplicates, 23 full text articles were reviewed, and 15 studies were eligible for final inclusion. Two reviewers assessed study quality and extracted data independently. Analysis of the extracted data was performed using 'Comprehensive Meta-Analysis.'

RESULTS

The 15 included studies compared outcomes of 6MWT, TUG, QTFA, and SF-36 between OI prostheses and socket prostheses. The analysis favored OI prostheses with large effect sizes of statistical significance for all noted outcomes; 6MWT (ES=2.576, P<0.001), TUG (ES=2.156, P<0.001), QTFA (ES=2.981, P<0.001), and SF-36 (ES=2.068, P<0.001) in LE amputees.

CONCLUSIONS OI prostheses were found to produce superior functional outcomes in both 6MWT and TUG as well as superior self-reported metrics in both QTFA and SF-36 scores in LE amputees.




A Review of Fall Detection and Fall Prediction Systems


Lorna Migiro

Kennesaw State University

BACKGROUND

Falls threaten seniors' safety, their independence and generate enormous economic and personal costs. Methodologies for anticipating falls are essential as the benefit of prevention far outweigh those of rehabilitation. There is a current surge of smart sensors and Internet of Things being incorporated in critical fall detection and prediction safety applications. However, in recent years, there is a lack of survey reviewing and discussing on these novel sensors, technologies and algorithms introduced and employed as well as the emerging challenges and new opportunities. This paper is a survey of the state of the art of technology-based fall detection and prediction systems, which are identified from a systematic review of studies presented in contemporary research literature. It aims to serve as a point of reference for future research on the mentioned systems

METHODS

The review of the current literature was performed to explore sources of literature from ACM, IEEE digital library, PubMed, web of science and science direct. We have performed searches using the keyword "fall detection", "fall prevention", "smart fall detection", "smart fall prevention". We limit the articles between year 2010 and 2020. We gave priority to papers whose intention was broad characterization of the tech - based fall detection and prediction systems

RESULTS

This review identified 41 projects that used wearable systems and 23 projects using non-wearable systems. Context aware and vision-based systems were found to be less popular with the elderly as they lack privacy. 90 % of approaches reviewed leverage on assimilating the smartphone sensor data in generating fall prediction machine learning algorithms. Multi sensor approaches that combine acceleration magnitude, sensor velocity, and body posture give the highest fall sensitivity and the lowest false positive rate

CONCLUSIONS Fall detection and prediction systems whether physiology or kinematics both rely on sensors and fall factors. With smart sensors and Internet of Things (IoT) developing rapidly, this field has made great progress. Whereas fall prediction systems are typically developed to estimate real-time or future fall risk, real time emulations among the elderly is difficult to obtain and all studies reviewed use young adults whose representation still remains controversial, thus poses challenge on how to put this studies into practice




A Portable Optoelectronic Device for Lung Disease Diagnosis Through an Exhaled Breath


Sanjay Sarma Oruganti Venkata

University of Georgia

BACKGROUND

Diagnosis of lung diseases in both adults and children begins with the physical observation of the patient's respiratory efforts and comparing them to the respiratory rate thresholds set forth by the WHO. Beyond breath rate identification, other diagnostic measures, including auscultation by stethoscope, cultures, blood serum examination, complete blood cell count, and chest radiography (x-rays). Regardless of the identification through these methods, they lack the capability of quickly and accurately identifying the infection-causing pathogen-a critical aspect to consider when deciding treatment course as there are a wide variety of treatments available.

METHODS

Considering the shortcomings in the current diagnostic procedures, we developed a highly portable online device for detecting the particle densities and pathogen composition in a breath. Our invention under progress is a highly portable optoelectronic device that detects a breath's optical scatter characteristics passed/ blown through it. To assimilate the breathing in humans, we conducted preliminary experiments by blowing aerosol particles into the device for detecting the particle density and flow characteristics.

CONCLUSIONS We further plan to conduct experiments with various bacteria types suspended in the aerosols similar to an exhaled breath. In the current poster, we introduce the device and the results obtained from our preliminary experiments.




Characterization of E-Cigarette Aerosol Deposition in the Human Lung Airways With a Comparison to Conventional Tobacco Cigarettes


Sara Spalding

School of Engineering, Mercer University

BACKGROUND

In recent years, electronic cigarettes (e-cigarettes) have been under scrutiny due to their adverse health effects, although they had been advertised as a healthier option for current smokers. The effects of e-cigarette flavors and various flow rates on aerosol particle concentrations were evaluated. Analysis of inhaled aerosol particles in human airways using analytical lung morphometry software was performed. Additionally, a comparison of aerosol characteristics of the e-cigarette and the conventional tobacco cigarette was performed.

METHODS

Aerosol characteristics from e-cigarettes and tobacco cigarettes were measured using a Wide Range Particle Spectrometer (WPS, 1 LPM), which can measure the concentration of aerosols in the range of 10 nm to 10 microns. The Multiple-Path Particle Dosimetry Model (MPPD) was used to calculate the amount of aerosol deposited in the lung airways.

RESULTS

The maximum particle number concentrations in ambient air, filtered air, and e-cigarette aerosols during the experiment were 57, 7, and 113,428 number of particles/cc, respectively. The influence of ambient and filtered air is negligible for particle concentration measurements. It was found that there is no statistical difference in the particle concentrations of different flavors and flow rates. The experimental data used in the MPPD analysis was that of the 5% nicotine, 1.7 LPM, mint e-cigarette. Within the MPPD analysis, we compared the Stochastic Model, Stochastic 25 bpm (lung disease), Stochastic 6 bpm (breathing impairment), Yeh Schum 5 Lobe, Age-Specific 14 years old, and Age-Specific 21 years old. In the pulmonary region Age 21 and Stochastic 6 bpm had higher particle deposition, Stochastic 25 bpm had lower particle deposition. In the whole lung, Age 21 and Stochastic 6 bpm models had higher particle deposition, Age 14 had lower particle deposition. It was found that 13.04% of the cigarette smoke was deposited in the lungs, of that amount 62.89% deposited in the pulmonary region. In comparison, 6.97% of E-cigarette smoke deposited in the lungs, of that amount 44.28% deposited in the pulmonary region.

CONCLUSIONS When comparing the different MPPD models, the amount of particles deposited by the e-cigarette aerosol is dependent on the age and breathing ability of the user. Young adults had higher deposition in the whole lung and the pulmonary region. In reference to the e-cigarette aerosol, the highest amount of particles were deposited in the pulmonary airways of the lungs where O2-CO2 exchange occurs. This potentially causes inflammation of the pulmonary airways, which would hinder breathing. When comparing conventional cigarette smoke to e-cigarette aerosol, the conventional cigarette smoke deposits more particles in the lungs than e-cigarettes.




Assessing Improvements in the Quality of Life of Parents of Children with Down Syndrome After Using MapHabit's Visual Mapping Software


Kaylin White

MapHabit Inc.

BACKGROUND

There is a growing body of evidence that assistive digital technology may improve the Quality of Life (QoL) of individuals with intellectual disabilities. The MapHabit System (MHS) is an example of such a technology for it provides visual-mapping, step-by-step cues containing photos and audio, to assist users in completing tasks. The MHS has previously been used amongst memory-impaired individuals and was shown to facilitate the accomplishment of their activities of daily living (ADLs) more independently and improve the overall quality of life. Given the MHS promotes independence, we decided to explore the potential benefits of the MHS for both individuals with developmental disability and their caregivers.

METHODS

To address this possibility, a focus group of 6 parents was provided the MHS's visual-mapping software to implement into the daily routines of their children with Down syndrome for a 4-week period. The children of the parents within this focus group were between the ages of 5 and 17 years old. Throughout the trial period, participants were interviewed on a weekly basis to assess progress with the MapHabit app and to assist with map building and customization. At the end of the 4-week period, the parents completed an 18 item QoL assessment and an assessment of user satisfaction.

RESULTS

Each of the parents included in the trial reported some positive change or much positive change in one or more of the in the following areas: Completed activities of daily living more quickly, increased independence, increased social interaction, decreased frustration, increased social engagement, and/or overall improved QoL. The net promotor scores, indicative of participant satisfaction, obtained showed that parents found using visual maps with their children was satisfying and that they would recommend the MHS to a friend or colleague.

CONCLUSIONS The positive findings reported in this proof-of-concept study is the first instance of the MHS being utilized by individuals with developmental disability. These initial findings demonstrate that the MHS addresses the need for a personalized low-risk, low-cost, readily available, and portable assistive technology tool that assists with independence amongst families with children with Down syndrome. Furthermore, the MHS may be applicable to a broader range of neurodiverse individuals across the lifespan.





 

Food and Nutrition

Activity in Rhodococcus rhodochrous after PEGylation


Palmer Amos Georgia State University BACKGROUND

Acute lymphoblastic leukemia (ALL) is a cancer with roughly 6000 new cases diagnosed annually in the US, with a higher prevalence of children (Inaba 2013, Pui 2008). In ALL bone marrow prioritizes the overproduction of incompetent lymphoblasts, disrupting the balance of blood cells, leading to lower red blood cell and platelet count. The disruption causes shortness of breath, easy bruising, and prolonged bleeding. The cancerous lymphoblasts are incapable of synthesizing the amino acid asparagine needed for cellular reproduction, and to reproduce asparagine must be taken up from the bloodstream. Aspariginase is an enzyme which converts asparagine into aspartic acid and ammonia, injections of asparagine into patients with ALL has been shown to slow progression, allowing other treatments to take effect. The current methods for obtaining asparaginase utilize gram-negative bacteria such as Escherichia coli and Erwinia chrysanthemi. The current process for obtaining asparaginase from E. coli and E. chrysanthemi. A buildup of endotoxins derived from the lipopolysaccharide (LPS) cell wall from E. coli, E. chrysanthemi and other gram-negative bacteria can contribute to septic shock. (Landesman 1978)
Rhodococcus rhodochrous (RR) DAP 96253 is a non-pathogenic gram-positive soil bacterium which exhibits asparaginase activity when induced with urea and cobalt during growth. The gram-positive bacteria possess less LPS and fewer endotoxins than gram negative bacteria. PEGylation is the process of adding a poly-ethylene glycol chain to a protein to create a hydrophobic bubble around the protein, and is widely used in pharmaceutical applications as a preservative and to prevent the immune response from prematurely dismantling the target protein.

METHODS Asparaginase purification was performed with dialysis followed by Ion-exchange chromatography. 2-picoline borane, a nontoxic reducing agent facilitates bonding via reductive amination. Using the method for detection of ammonia developed by Fawcett and Scott CONCLUSIONS We are able to show asparaginase activity after PEGylation a week after PEGylation.




Detection of SARS-CoV-2 in the Environment


Jessica Bowman

Georgia Institute of Technology

BACKGROUND

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a severe and global impact. Identification of asymptomatic yet contagious individuals is key to controlling spread of the virus; however, testing each individual with frequency is resource demanding and logistically challenging. SARS-CoV-2 can be deposited to the environment from contagious individuals through coughing, sneezing, touching and other mechanisms. The half-life of viable SARS-CoV-2 in aerosols and on common surfaces has been demonstrated sufficient to transmit the disease.

METHODS We approached the issue of environmental risk by developing methods for collection and detection of SARS-CoV-2 nucleic acids from air and surfaces. We used swabbing techniques to collect on surfaces, and are testing multiple apparatus, including filtration and liquid impingement, to collect particles from air. Once collected and treated, environmental samples were tested for SARS-CoV-2 with the same sensitive and specific RT-qPCR technique used for diagnosis of potentially infected individuals. RESULTS

We identified cheap, effective, available and reliable swabs for collection of SARS-CoV-2 nucleic acids from surfaces. We also identified a suitable medium for wetting the swab prior to sample collection, and a method for lysing the virus and protecting viral RNA from degradation that did not interfere with downstream RT-qPCR. A slight modification on the RT-qPCR technique was used to differentiate wild viral RNA from environmental contamination by SARS-CoV-2 test materials. Standard curves generated with heat-inactivated virus, or SARS-CoV-2 test materials, could be used to quantitatively estimate the amount of SARS-CoV-2 nucleic acid found in the environment. Apparatus and methods for collection and detection of virus from air with similar techniques are feasible.

CONCLUSIONS Air and surfaces can be monitored for SARS-CoV-2 nucleic acids. Though the presence of nucleic acids does not necessarily indicate infection risk, their presence indicates the presence of potentially infective particles. Environmental monitoring for SARS-CoV-2 using RT-qPCR is sensitive and quantitative, and can be used to understand and control risk in public spaces.




DNA De-methylation Early in Life Underlies Sex Differences in Estrogen Receptor Alpha Expression in Specific Hypothalamic Areas


Iagn Nino Kenji Cabahug

Georgia State University

BACKGROUND

Neural sex differences may entail differential gene expression leading to differences in cell phenotype. For example, male rodents have more calbindin-expressing neurons (CALB) in the sexually dimorphic nucleus of the preoptic area (SDN-POA) than do females, whereas females have a greater number of neurons expressing estrogen receptor alpha (ERa) in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and the arcuate nucleus (ARC). Recent studies in our lab (Mosley et al., 2017; Cisternas, Cortes et al., 2020) suggest that DNA methylation is an important mechanism underlying sexual differentiation of cell phenotype in the brain. DNA methylation is catalyzed by DNA methyl transferases, enzymes which add methyl marks onto mainly cytosine residues. Conversely, DNA de-methylation is catalyzed by ten-eleven translocation (TETs) methylcytosine dioxygenases, which remove these methyl marks through a series of steps. Expression of both enzyme groups peaks early in life in the brain (Cisternas et al., 2020), suggesting a crucial role of epigenetics in neural development. In support of this, we demonstrated that early life inhibition of DNA methylation has lasting effects on CALB and ERa expression in the POA and VMHvl, leading to reduction or elimination of sex differences in adulthood (Mosly et. al, 2019). To our knowledge, no one has tested the role of TET enzymes in establishing sex differences in the brain. We hypothesized that the removal of DNA methyl marks is an equally important process of sexual differentiation of cell phenotype and predicted that interfering with TET enzyme expression early in life would disrupt sex differences in ERa labeling.

METHODS We injected small interfering RNA (siRNA; 400-500 pmol) targeting Tet2 and Tet3 via intracerebroventricular injections into postnatal day (P) 5 male pups. We chose this age and sex because males express higher levels of Tet2 and Tet3 relative to females in the hypothalamus on P7, and it takes ~48 hours for siRNA to reduce expression. We injected non-targeting siRNA as a control in same-age male and female pups. We collected brain tissue at P25 and performed immunohistochemistry against ERa. Labeling of ERa in the ARC and VMHvl was compared across groups using ImageJ, and statistical analyses were conducted using RStudio. RESULTS

One-way ANOVA revealed a significant difference between control males, siRNA-treated males and control females in ERa labeling in the ARC (F (2,20) = 4.662, p = 0.0218). As expected, females had more ERa labeling than males, although only marginally significant (p = 0.053). ERa labeling was increased to female levels in siRNA-treated males compared to control-males, (p = 0.038). In contrast, there was no effect of siRNA treatment in the neighboring region, the VMHvl, which also has a sex difference in ERa expression.

CONCLUSIONS We found that siRNA-mediated degradation of TET mRNA through ICV treatment on P5 led to a robust down-regulation of Tet2 and Tet3 expression in the hypothalamus. Furthermore, this reduction in Tet2 and Tet3 expression had a significant effect on ERa labeling in the ARC, but not in the VMHvl. This suggest region-specific modulation of gene expression via DNA demethylation.




Perigestational Exposure to Morphine Leads to Immune Deficits in Male and Female Rats


Hannah Harder

Georgia State University

BACKGROUND

Neonatal opioid withdrawal syndrome, or NOWS, occurs when women use opioids during pregnancy, causing the fetus to become dependent on opioids. At birth, the child is removed from the opioid source and experiences withdrawal symptoms, including autonomic dysfunction, hypersensitivity, and gastrointestinal distress. Clinical chart reviews have associated NOWS with an increased risk of hospitalization with infections, and preclinical evidence suggests opioids are immunosuppressive in adult animals. Therefore, we hypothesize that perigestational opioid exposure leads to long-term immune dysfunction.

METHODS Female Sprague-Dawley rats at P60 are implanted with iPrecio® microinfusion pumps, and given 10 mg/kg morphine/day in 3 doses. The following week, animals are bred to induce pregnancy and the dose is increased to 12 mg/kg/day, and further increased weekly to 14 and 16 mg/kg/day. At E18, doses decrease from 3 to 2 times a day. At birth, dams continue to receive morphine, and pass it to their pups through milk. When the pups reach P5, the dams' morphine dose is slowly decreased, dropping to 0 mg/kg/day by P7. This model represents the clinical features of maternal drug use in humans, including pregestational, gestational, and post-partum drug use, pulsatile dosing, and dose modulation throughout pregnancy. Pups receive indirect morphine throughout gestation and early development during critical periods of brain and immune system development. As adults, morphine-exposed male and female offspring are implanted with temperature loggers and given an immune challenge of lipopolysaccharide, which induces a characteristic fever and sickness behavior response. Fever and sickness behavior is measured for six hours post-LPS injection. Sickness behavior is scored from 0 to 8, with larger numbers representing more sick animals. RESULTS

Physiological data from morphine-exposed pups shows minor differences in weight during early life (~10-20% lower body weight). Morphine-exposed males showed a hypothermic response to LPS, as opposed to the typical hyperthermic response seen in vehicle-exposed males and females. Vehicle-exposed males and females had a net temperature change over six hours of approximately 2-3 ℃, while males had a net temperature change of -1 ℃. Interestingly, morphine-exposed females showed a net temperature change of 0 ℃, suggesting an equal hypothermic and hyperthermic response. Sickness behavior was equal between morphine- and vehicle-exposed males, while morphine-exposed females show increased sickness behavior vs. vehicle-exposed females (2 vs 1 on the sickness scale).

CONCLUSIONS We conclude that morphine-exposed male and female pups show altered immune function, both fever and sickness behavior, after LPS challenge, and that these effects are sex-dependent with morphine-exposed males potentially showing a more severe deficit. Opioid exposure during gestation likely creates long-term immune deficits due to developmental changes in the immune system, suggesting that infants born with NOWS may be at risk for immunosuppression.




Empagliflozin relaxes systemic mesenteric arteries via the activation of voltage-dependent K+ (KV) channels in arterial smooth muscle cells


Ahasanul Hasan

Mercer University

BACKGROUND

Empagliflozin, a sodium glucose cotransporter-2 (SGLT2) inhibitor, belongs to a new class of antidiabetic drugs that, in addition to reducing glucose reabsorption through kidneys, produces favorable cardiovascular effects, including a reduction of blood pressure. However, it is not clear if empagliflozin directly regulates contractility of resistance mesenteric arteries. We therefore sought to investigate direct vascular effects on empagliflozin on resistance mesenteric arteries and underlying molecular mechanisms.

METHODS To examine if empagliflozin regulates arterial contractility, we performed pressurized arterial myography on freshly isolated, ex vivo preparation of resistance-size (150-300µm), mesenteric arteries from 6-9 weeks old Sprague Dawley rats. To study the role of endothelial signaling, endothelium-intact and -denuded mesenteric arteries were used. Endothelium was denuded by slow passage of air bubbles through the vessel lumen. Absence or at least 70% reduction of acetylcholine-induced vasodilation was used as the indicator of endothelium denudation. RESULTS

Our pressure myography data showed that empagliflozin produced a concentration-dependent (0.001-100µM) vasodilation of myogenic arteries (5.76%-13.30%, expressed as % of maximum passive diameter at 80mmHg) and phenylephrine (PE)-preconstricted arteries (3.69%-72.24%, expressed as % inhibition of PE-induced contraction). To understand the mechanism(s) underlying empagliflozin-induced vasodilation, we assessed the contribution of endothelium- and smooth muscle-specific vasodilatory signaling pathways. We first investigated the role of the endothelium in empagliflozin-evoked vasorelaxation by comparing responses of endothelium-intact- and -denuded mesenteric arteries. Our data demonstrate that the magnitude of empagliflozin-induced vasodilation was similar in endothelium-intact (51.91±1.87%) and -denuded (48.81±1.87%) arteries. We further examined the role of nitric oxide (NO)-guanylyl cyclase (GC)-protein kinase G (PKG) signaling axis. Consistently, pharmacological inhibition of eNOS with L-NAME did not alter empagliflozin-evoked vasodilation (96 ± 2.62% vs 89.5 ± 6.91%). Inhibition of GC with ODQ (34.75 ± 7.90% vs 35.43 ± 4.39%) or the inhibition of PKG with KT5823 (46.53±4.23% vs 49.70 ± 8.06%) had no significant effects on empagliflozin-induced vasodilation. We next examined the role of smooth muscle cell K+ channels that control membrane potential and arterial contractility. We found that the inhibition of KV channels with 4-aminopyridine significantly reduced empagliflozin-induced vasodilation (80.37±7.47% vs 24.92±2.92%). However, inhibition of BKCa channels with paxilline, inhibition of KATP channels with glibenclamide or the inhibition of cyclooxygenase enzyme with indomethacin were without significant effects. These data suggest that smooth muscle cell KV channels are essential for mediating vasodilatory effects of empagliflozin.

CONCLUSIONS Overall, our data suggest that empagliflozin produces mesenteric artery vasodilation via the activation smooth muscle cells KV channels, without the involvement of endothelial signaling. Future studies will investigate if empagliflozin-induced arterial vasodilation can lower systemic blood pressure in healthy and in diabetic animal models.




Diverse Roles of G-Protein-Coupled Receptor 40 (GPR40/FFA1) and GPR120 (FFA4) in the Regulation of Cell Growth and Motile Activities in Renal Cell Carcinoma (RCC)


Priyanka Florina Karmokar

Mercer University

BACKGROUND

Renal cell carcinoma (RCC) is the 16th most common cancer worldwide with roughly 400,000 new cases diagnosed annually. Even though the 5-year survival rate for early-stage RCC is 80%-90%, it is only 5% for the patients with metastasis. Unfortunately, anticancer drugs that are used to treat RCC are proven to be useful for only a small number of patients. Therefore, it is crucial to discover the novel molecular pathways underlying RCC to develop better therapies. The G-protein-coupled receptors GPR40 and GPR120, also referred to as free fatty acid receptor 1 (FFA1) and 4 (FFA4), respectively, are activated by medium- to long-chain free fatty acids. Emerging evidence suggests that GPR40 and GPR120 are implicated in the proliferation, invasiveness, and apoptosis of various tumor cells, however, the precise mechanisms of GPCRs signaling events leading to the modifications in cancer cell growth and migration are still scarce, and the roles of these receptors in RCC are unstudied to date. Here, for the first time, we reveal the effects of GPR40 and GPR120 agonism on cell proliferation and migration in metastatic tumor-derived renal cancer cells.

METHODS Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to confirm the presence of GPR40 and GPR120 transcripts. Cell proliferation was assessed by plating 50,000 cells per well, followed by treatment with GPR40 and GPR120 stimulants as noted for 6 days. Cells were counted every 24 hours. For wound-healing assay, 100,000 cells were plated on a 48-well plate and a scratch made through the center. The time to wound healing was assessed at 24, 36, and 48 hr. For migration assays, cells were plated in the top chamber of a transwell plate and cells migrated was counted by crystal violet staining after 16 hr. RESULTS

Using selective GPR40 and GPR120 agonists and antagonists, our results demonstrate that GPR40 enhanced and GPR120 suppressed cell proliferation, suggesting that the opposite effects of GPR120 and GPR40 are involved in the cell growth activity. The wound-healing assay revealed that GPR120 agonism markedly covered the wounded area compared to vehicle-controls, an effect that is blocked by a selective GPR120 antagonist. However, GPR40 agonism demonstrates no significant effect, suggesting that GPR40 may not contribute to the whole-cell mass migration. In transwell migration assay, both GPR40 and GPR120 agonists elevated the ability of single cells to respond to the chemoattractant compared to vehicle-control, an effect reversed by potent GPR40 and GPR120 antagonists, respectively; further confirming their involvement in the acquisition of migratory properties in RCC cells.

CONCLUSIONS Our present results suggest that GPR40/120-mediated signaling may be involved in the regulation of renal cancer tumorigenesis and progression on the cellular level, and mechanistic studies are underway to assess the cellular cascades that are influenced by GPR40 and GPR120.




Auxin stimulates vasodilation of resistance mesenteric arteries


Sreelakshmi Menon

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Accumulating evidence suggests that short-chain fatty acids synthesized in the human body by the action of gut microbiota on the dietary fibers by anaerobic fermentation have beneficial effects on the cardiovascular system. Here, we examined the effects of auxin, a short-chain fatty acid and a plant growth hormone found in vegetable stem tips, on the contractility of Sprague Dawlay rat mesenteric arteries using pressurized artery myography and tissue nitric oxide (NO) measurement.

METHODS Fresh isolated fourth order rat mesenteric arteries were cannulated and maintained in a temperature-controlled perfusion chamber. Arterial segments were subjected to a stepwise increase in intraluminal pressure to 80 mmHg. Development of sustained myogenic tone at 80 mmHg generated a baseline diameter after which increasing concentrations (1nM to 1mM) of auxin were applied and diameter changes tracked. Quantification of tissue NO was performed after treating the tissue with auxin. RESULTS

Our pressurized artery myography data showed that auxin produced a concentration-dependent vasodilation of mesenteric arteries, with a maximum dilation of 27 µm at 1 mM concentration. To understand the mechanism underlying auxin-induced vasodilation, we next investigated the role of endothelial NO production. Our data showed that co-application of 10 µM L-NNA, a selective inhibitor of endothelial nitric oxide synthase (eNOS), caused 100% reversal of 1 mM auxin-evoked vasodilation of mesenteric arteries. Consistent with our pressure myography data, quantification of tissue NO also demonstrated that the treatment of mesenteric arteries with auxin indeed enhanced NO production.

CONCLUSIONS

Altogether, our data unveils a novel role for auxin in producing systemic mesenteric artery vasodilation via endothelial NO production. Future studies will investigate the detailed mechanism for auxin-mediated mesenteric artery vasodilation and its relevance for systemic blood pressure regulation.




Microelectrode Sensors for MicroRNAs and COVID-19 Viral RNAs


Jonathan Padelford

Georgia State University

BACKGROUND

Rapid and accurate detection of RNA species such as microRNAs and viral RNAs is increasing essential especially with the COVID-19 pandemic due to the SARS-CoV2 coronavirus. Current fast testing tools suffer significantly from the high false negative/positive rates. Laboratory methods such as real-time polymerase chain reaction (RT-PCR) are time and resource extensive, and the analysis accuracy heavily rely on trained personnel and experience due to their multistep sample treatments. Our one-step label-free electrochemical technique developed for the quantitation of microRNAs is being redesigned for the analysis of SARS-CoV2 viral RNAs and is both highly sensitive and selective.

METHODS Gold microdisk electrodes are fabricated via benchtop methods. The radii of the Au microelectrodes are in the range of several microns, sub-micron and tens of microns. A multi-segment recognition probe, which includes a nucleic acid sequence that perfectly complement the target RNAs, will be covalently attached to the Au microelectrodes via Au-thiol bonding. Several complementary sequences to the viral RNA sequences used in RT-PCR for the detection of SARS-CoV2 are chosen given their comparable length to microRNAs (and thus affinity). The tool development and target quantitation are achieved with an array of electroanalytical methods notably cyclic voltammetry (CV) and square wave voltammetry (SWV). RESULTS

This approach is supported by our previous successes to develop sensors for the microRNA miR-122. A lower limit of detection at 0.1 femtomolar (fM) and the dynamic range up to 0.5 nanomolar (nM) has been demonstrated with the introduced signal amplification mechanism and a pristine negligible background. Importantly, the detection is one-step and completes within minutes allowing for not only an extremely low level of detection but also a quick turnaround time. The quantification in absolute abundance of the target RNAs can be achieved using the electrochemical readout which is significant for both biomedical research as well as point-of-care usage. The sensors also showed superior specificity in the discrimination against analogs such as two-base mismatches of miR-122.

CONCLUSIONS This presentation explains the design principles of signal-on electrochemical sensors for 20-30 base RNAs, for example microRNAs and the viral RNAs demonstrated to be effective in RT-PCR analysis. The one-step detection of sub-femto-molar target (microRNA-122) within minutes has been achieved which is adequate for the microRNA analysis under physiological relevant range. The versatile sensor design make it readily repurposed to target other microRNAs or viral RNAs. Additional DNA-based probes targeting COVID-19 primers have been designed and sensor development is underway. Ultimately, our goal is to offer a capable tool to alleviate the immediate testing problems related to COVID-19 and be prepared for the future (mutations or other RNA viruses).




Regulation of Arterial Contractility by Neflamapimod (VX-745), an investigational drug for Alzheimer's disease


Ajay Pandey

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Alzheimer's disease is the 6th leading cause of death in the United States affecting approximately 5 million people. Preclinical studies and data from a recent phase II clinical trial suggest that neflamapimod (previously code named VX-745), a p38α MAP kinase inhibitor, reduces inflammation and reverses cognitive deficits in Alzheimer's disease. Since vascular dysfunction is linked to neurodegeneration and cognitive dysfunction in Alzheimer's disease, candidate drugs that have inherent vasodilatory effects are likely to have positive impacts on the disease outcome. Therefore, we sought to investigate the direct vascular effects of neflamapimod and characterize underlying mechanisms.

METHODS Using pressurized arterial myography and calcium imaging, we sought to investigate the direct vascular effects of therapeutic concentrations of neflamapimod on resistance mesenteric and cerebral arteries from Sprague Dawley rats. Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressured gradually increased to 80 mmHg. After sustained development myogenic tone at 80 mmHg, neflamapimod(100pM to 10μM) and pharmacological modulators were applied, and diameter changes tracked. For calcium imaging, the isolated arteries were preincubated in Fura-2AM(10µM) and 0.02% Kolliphor solution at room temperature for two hours before being mounted in the perfusion chamber. RESULTS

Our pressure myography data showed that neflamapimod produced a dose-dependent vasodilation of mesenteric arteries, with 1 and 10μM concentrations producing ~16μm and ~37μm dilation, respectively. Such vasodilation was also seen in cerebral arteries, however, to a lesser extent (~11μm and ~17μm dilation by 1 and 10μM concentrations). To delineate the mechanism underlying neflamapimod-induced vasodilation, we assessed the contribution of endothelium- and smooth muscle-specific vasodilatory signaling pathways. The vasodilatory responses produced by the highest concentration in endothelium-intact and -denuded arteries were ~37μm and ~39μm, respectively, suggesting that the mechanism is endothelium-independent. Consistently, inhibition of endothelium-derived vasodilatory pathways, such as those mediated by endothelial nitric oxide synthase (eNOS) and PGI2 had no effect on the vasodilation either. We next examined the role of smooth muscle cell K+ channels. Our data demonstrated that neflamapimod-evoked vasodilation of resistance mesenteric arteries was unaltered by the inhibition of smooth muscle cell K+ channels such as large-conductance Ca2+-activated potassium channels (BKCa: ~36μm versus ~37μm), ATP-sensitive potassium channels (KATP: ~53μm versus ~37μm), and voltage-gated potassium channels (KV: ~28μm versus ~37μm). Overall, our data suggest that neflamapimod produces vasodilation via a smooth muscle-specific mechanism that remains to be determined.

CONCLUSIONS We found that neflamapimod produces a dose-dependent vasodilation of both mesenteric and cerebral arteries that does not depend on endothelium-derived vasodilatory mechanisms or smooth muscle potassium channels. Future studies will examine the role of PKA, PKC, Rho-kinase and MAP kinase signaling in mediating neflamapimod-evoked vasodilation of cerebral and mesenteric arteries.




Polarimetric Second Harmonic Generation can be Performed in Full-thickness Bone and is Useful in Discriminating Between Healthy and Diseased States


Emily Pendleton

University of Georgia

BACKGROUND

Bone is a unique biological composite material made up of a highly structured collagen mesh matrix and mineral deposits. Although mineral provides stiffness, collagen's secondary organization provides a critical role in bone elasticity. Together, these biological components work together to form healthy bone and a disruption in one of these elements causes deleterious effects to the formation of the other. Yet, we currently only image and assess bone health with bone mineral density. Here, we work to develop a method to analyze collagen structure in full-thickness bone and implement this method to distinguish healthy bone from diseased bone.

METHODS

Our optical setup includes a Calmar fiber laser producing 1550 nm 370 fs pulses of 3uJ energy with power modulated with a half wave-plate. The polarization angle was rotated using another half wave-plate and second harmonic signal of collagen was collected. Using established theories, we determined the angle of the collagen structures within each image. To develop this method, we used three adult mouse skulls. To analyze the difference of the collagen structure, we used 13 healthy mice and 13 mice afflicted with Hypophosphatasia (HPP), a metabolic bone disorder which inhibits mineralization. To evaluate these images, we established a novel method of integrating polarimetric images with gray-level co-occurrence matrix (GLCM) texture analysis.

RESULTS

We performed polarimetric analysis of bone collagen fibers in adult mouse skulls using second harmonic generation (SHG) imaging to evaluate lamella sheets and collagen fiber integrity in intact cranial bone. Our polarimetric data was fitted to a model accounting for diattenuation, polarization cross-talk, and birefringence. We compared our data to the fitted model and found no significant difference between our polarimetric observation and the representation of these scattering properties up to 70 µm deep. We also observed a loss of resolution as we imaged up to 70 µm deep into bone but a conservation of polarimetric response.

Once this method was established, we used it to describe collagen fiber and lamella sheet orientation as well as anisotropic values in both healthy and HPP mice. We developed a novel method of angular GLCM analysis and were able to separate these two groups with PLS-DA analysis with 0.00% misclassification rate using the leave-one-out method.

CONCLUSIONS We established the analytical use of polarimetric SHG which allows for the discrimination of collagen lamellar sheet structures in intact bone. We then used this work to describe collagen defects for the first time in HPP. Furthermore, we developed a novel angular GLCM technique that effectively separated healthy from diseased bone. Our work could allow for label-free identification of disease states and monitor the efficacy of therapies for bone disorders.




Community-level Analyses of Anthropogenically Afflicted Jamaica Bay, NY Using the 16S rRNA Biomarker


Fortino Pineda Veloz

Georgia State University

BACKGROUND

Jamaica Bay, adjacent to New York City (NYC), has been afflicted by nitrogen saturation due to urban runoff and an outdated combined sewer system; in addition to physical alterations from urban development and demand. This has resulted in recurring harmful algal blooms (HABs) and an increase in average low tide depth with 85% of the wetland area lost in the past 150 years. In the past decade, the NYC Department of Environmental Protection has allotted $520 million dollars to mitigate anthropogenic effects on the Jamaica Bay estuary and restore this harbor for flora and fauna. This study describes the microbial community structure from one-liter surface water samples taken in 2016 and implements functional prediction to ascertain activity in eutrophic waters

METHODS

The Citizens Water Quality Testing (CWQT) group collected one-liter surface grab samples at three distinct locations: Canarsie Pier (high human activity), Floyd Bennett Field (low human activity), and Paedergat Basin (Combined Sewer Overflow site). Samples had DNA extracted and went through the Illumina MiSeq sequencing by synthesis protocol to generate forward single end reads of the V3/V4 region of the 16S rRNA gene.
Downstream analysis consisted of the Quantitative Insights Into Microbial Ecology version 2 (QIIME2) using amplicon correction and quality filtering with the Divisive Amplicon Denoising Algorithm 2 (DADA2). 94,000 filtered sequences from each sample were taxonomically classified using the SILVA 128 rRNA database comprising of environmental samples. The diversity and richness within the samples were adequately represented in the Shannon index and Faith PD Plot. Sampling date and temperature were used to generate a Principle Coordinates Plot (PCoA) to visualize phylogenetic differences in cluster. The statistical relevance of each factor was quantified with p-values using a PERMANOVA analysis with 999 permutations.
The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) functional abundance prediction pipeline employed the DADA2 16S rRNA gene marker data alongside feature counts to construct a quantitative scope of gene families within the samples. Differences in functional profiles were predicted and recorded.

RESULTS

The PERMANOVA analysis found no correlation between sampling site with a p-value of 0.989, while there was significant correlation when sorting by sampling date with a p-value of 0.008 (p < 0.01). The climate over 6 days of each sampling date was investigated to create the following chart.

CONCLUSIONS

The most consist temperature over a six-day period was on August 18th, 2016 which showed congruence to the PCoA plot clustering. In addition, the most varied period was that of July 28th, 2016 which reflect the spread of its respective data points in the PCoA plot. These relations were also seen in the taxonomic profiling of each sample with a highly consistent profile belong to those samples taken on August 18th, 2016 as well as the inverse in the case of samples taken on July 28th, 2016.
Cyanobacteria dominate the community with values ranging from 30% to 40%. The secondmost dominant taxa was Proteobacteria, led by the subgroup Alphaproteobactertia then Gammaproteobacteria. The latter has been seen to be associated with shifts in water temperature within Cheasapeake Bay. The relation between these subgroups of Proteobacteria and temperature may be confounded by a synergistic companinionship with Cyanobacteria. The former degrades Dissolved Organic Nitrogen (DON) in the form of nitrates to produce Dissolved Inorganic Nitrogen (DIN), fostering phytoplankton booms. The many years of nitrogen-rich effluent have maintained this relationship and created a loss of microbial diversity. Furthermore, the microbial community is affected as the termination of phytoplankton blooms create anoxic regions in the bay via respiration of sinking biomass. Some bacteriaplankton also possess the ability to cycle carbon using nitrate in anoxic region-leading to the recurrence of blooms. This was also respresented functionally with support in photosynthetic genes.
Temperature increases the kinetics of reactions and creates a bias in metabolic activity. A more all encompassing chart is in design and will be ready. It will include all sampling organized by date rather than the metagenomic data created in KRAKEN as seen above.
In conclusion, the physical properties of Jamaica Bay and its community structure do not align well with the diversity-stability hypothesis. This hypothesis states that a diverse community is better suited to withstand destabilizing events. However, Jamaica Bay no longer reflects other estuaries diversity due to its prolonged infliction with nitrogen-loading, leading to a community built on a nutritive bias. As climate change continues and sea levels rise, one cannot be sure of the progress of restoration nor of the half billion dollar NYC initiative without increased sampling efforts. In this unique complex estuarine system, additional data must be generated to statistically correlate temporal changes within the bay's ecosystem to environmental factors in the form of a longitudinal study with autonomous abiotic sampling.




Molecular Identification and Characterization of Nitrile Hydratase in Rhodococcus rhodochrous DAP 96253


Fortino Pineda Veloz

Georgia State University

BACKGROUND

Nitrile hydratase (NHase) is an enzyme that hydrolyzes nitrile moieties into their corresponding amide form. When NHase catalyzes a reaction with the substrate acrylonitrile, acrylamide is formed. Acrylamide is the building block of polyacrylamide (PAM); a polymer used for coagulation during water treatment, gel electrophoresis chambers, and various manufactured polymers. The interest in this strain's enzymatic capability stems from the industrial leader in biotransformation--Rhodococcus rhodochrous J1. R. rhodochrous J1 is used to produce over 30,000 tons of acrylamide annually. It possesses high caliber enzymatic ability with percent yields hovering at 99% whilst bypassing acrylic acid as an unwanted byproduct.

R. rhodochrous DAP 96253 cells have been induced with urea and cobalt to increase nitrile hydratase activity which benefits the industrial upside of this strain. Low molecular weight nitrile hydratase (L-NHase) catalyzes the reaction first. Upon sensing the amide reaction product, high molecular weight nitrile hydratase (H-NHase) is induced, increasing catalytic activity of acrylonitrile. By gaining knowledge of transcriptional regulation, NHase production has the potential to be upregulated beyond its current level. In addition, understanding the genetic construct of R. rhodochrous DAP 96253 is paramount to further molecular and in-silico studies on the enzyme of interest.

METHODS

Sterile Eppendorf tubes containing 50 mg of glucose-urea-cobalt induced R. rhodochrous DAP 96253 cell paste were refrigerated for 45 minutes at -20°C. The paste was suspended in 250 μL of lysozyme (10 mg/mL) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 20 μL of mutanolysin (1 mg/mL). The mixture was incubated for an hour at 37°C with each tube gently shook every ten minutes. Following incubation, 50 μL of 0.5 M EDTA, 4M NaCl, and 10% SDS were added. Proteinase K (20 mg/mL) was added in the volume of 10 μL to each tube then incubated at 37°C for one hour. 350 μL of this mixture were extracted and mixed with one volume of phenol:chloroform:isoamyl alcohol (25:24:1). The tubes were then centrifuged at 13,000 x g for five minutes. The top aqueous layer had 300 microliters extracted and underwent liquid to liquid extraction with a half volume of chloroform:isoamyl alcohol. The solution was inverted several times and centrifuged at 13,000 x g for five minutes. The aqueous layer was again extracted, and precipitation followed with one volume of chilled isopropanol. The sample spun at 13,000 x g for 15 minutes. The resulting pellet was washed with 200 μL of 70% ethanol, centrifuged at 13,000 x g for 10 minutes, and resuspended in 100 μL of TE buffer. Quantification of yield and purity were conducted using a Thermo Scientific Spectrophotometer Nanodrop 2000C.

Genomic DNA of R.rhodococcus DAP 96253 was sequenced via the Ion Torrent Xpress Personal Genome machine through reconstruction of fragmented DNA. The outputted file contained possible protein products that were compared to the NCBI Basic Local Alignment Search Tool (BLAST) for protein sequences. The order and location (positive or negative strand) of those proteins identical or similar to NHase genes of the Rhodococcus genus were organized into Figure 2. Nucleotide sequences from the Ion Xpress output file were used to construct forward and reverse primers for future PCR. Primers will contain restriction enzyme recognition sites to ligate the nucleotide splice into a plasmid vector under a lactose operon for characterization of the expressed protein with subsequent assays.

CONCLUSIONS

A genetic framework for nitrile hydratase been established for the first time with R. rhodochrous DAP 96253. Future molecular work will confirm nitrile hydratase through ligation of this genetic splice into a plasmid vector. The vector will allow for control of expression under a lactose operon. Verification of analogous transcription factors of the DAP 96253 strain to the J1 strain will be done via knock out studies. Interestingly, there was no discovery of nnhE in R. rhodochrous DAP 96253 when analyzing the fragmented genomic library. This protein has no known function and it will be imperative to investigate if this protein is present in an alternate sequencing method as well as its role in H-NHase.




Inclusion of Ischemic Stroke Lesion Topology in a Translational Porcine Model Leads to Better Prediction of Functional Outcomes


Kelly Scheulin

University of Georgia

BACKGROUND

Predicting stroke patient functional outcomes based on acute magnetic resonance imaging (MRI) information has significant clinical value in determining prognosis and potential course of treatment. The inclusion of stroke lesion location when predicting functional outcomes is likely to improve prognostic ability as there is a direct relationship between brain structures and function. However, stroke location is under assessed in predicting functional outcomes with traditional metrics such as lesion volumes and midline shifts being more heavily used pre-clinically and clinically.

METHODS

The objective of this current study is to identify lesioned brain structures by MRI and correlate structural alterations with changes in motor function and behavior in a preclinical porcine model. Pigs (n=7) underwent stroke surgery where the middle cerebral artery was permanently occluded (MCAO). MRI was performed at 1 day (d) post-MCAO and functional gait analysis and open field testing were performed on 0d, 2d, 8d, 15d, and 27d post-MCAO. A porcine MRI brain atlas was registered to identify stroke lesion location, and linear regressions between infarcted brain structures and functional data were completed to evaluate the predictive capacity of individual brain structure lesion on neurological outcome.

RESULTS

MCAO resulted in prominent lesion volumes and decreased white matter integrity. Highly lesioned brain structures included the insular cortex, somatosensory cortices, visual cortices, temporal gyri, and putamen. MCAO severely impaired translational gait parameters, decreased voluntary movement in open field testing, and resulted in increased modified Rankin Scale (mRS) scoring. Linear regression analysis determined that lesions in the secondary visual cortex, claustrum, amygdala, and superior temporal gyrus were highly prognostic of overall gait and behavioral outcomes.

CONCLUSIONS

This regression analysis approach identified neuroanatomical structures that were predictive of stroke outcome and identified potential target structures for therapeutic intervention to facilitate optimal functional recovery.




Differential Modulation of β2AR Mediated cAMP/PKA/CREB Signaling Cascade in Normal versus Asthmatic Airway Epithelial Cells


Kirti Singh

Graduate Student, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University

BACKGROUND

Nearly 25 million people in the U.S. suffer from asthma. While there is no panacea for asthma, symptomatic relief is achieved by the current therapeutic interventions, including β2-adrenergic receptor (β2AR) agonists. Chronic usage of β2AR agonists can have serious implications like airway hyper-responsiveness and receptor desensitization, and along with unknown mechanisms, can ultimately culminate into tachyphylaxis to the response. Previously, our laboratory has extensively explored the critical role of reactive oxygen species (ROS) in modulating β2AR function. To begin to assess the physiological role of the β2AR-ROS linkage in asthma, here, we assessed Isoproterenol (ISO)-induced β2AR mediated Gαs/cAMP/CREB pathway in small airway epithelial cells from asthma patients (SAEC-A) compared to those from non-diseased control patients (SAEC-N). Given the importance of β2AR mediated cAMP production and its role in airway bronchodilation, as well as tachyphylaxis that occurs upon chronic use of β2AR agonists, our results may shed light on the role of oxidants in altering β2AR responses in asthma.

METHODS

To assess cAMP levels, cells were analyzed using highly sensitive luciferase-based biosensor. Briefly, SAEC were transfected with GloSensorTM -22F cAMP plasmid (8µg) using Lipofectamine3000. Luminescence was measured following treated with the ISO. To assess the effects of downstream cAMP-mediated transcription, cells were stimulated with H2O, Forskolin (FSK) (10 µM) & ISO (100µM) for 20 min and lysed. Lysates were resolved using SDS-PAGE and immunoprobed for phospho-cAMP response element-binding protein (pCREB), total CREB and β-actin. Similarly, SAEC-N/A lysates were assessed for Adenylyl Cyclase (AC-5/6) and Phosphodiesterase (PDE4) by immunoblot.

RESULTS

(1) β2AR agonism with ISO significantly increased cAMP production in SAEC from asthma patients compared to non-diseased control. (2) In spite of elevated cAMP production in asthmatic SAEC, both ISO and FSK were able to induce significant increase in CREB phosphorylation, leading to heightened pCREB/CREB response in SAEC-N as compared to SAEC-A. (3) ISO-mediated increased cAMP response in asthmatic SAEC compared to non-diseased control, were linked with physiologically altered expression of key cAMP mediators, specifically, heightened basal expression of AC-5/6 and PDE4, in asthmatic SAEC compared to SAEC-N.

CONCLUSIONS

ISO mediated increased cAMP production in asthmatic SAEC indicated altered agonist modulated β2AR downstream signaling in diseased versus non-diseased SAEC. Further, heightened basal expression of AC-5/6 in asthmatic SAEC supports the above mentioned consequence. In addition, elevated CREB phosphorylation in non-diseased SAEC suggests alterations of β2AR-mediated PKA/CREB signaling in asthma. Future results will probe the effects of elevated ROS in these signal cascades.




Hypoxia-Inducible Factor 1-α (HIF-1α) Regulates Alzheimer's Disease-Like Neuropathology during Thiamine Insufficiency


Maria Valle

University of Georgia

BACKGROUND

Thiamine is an essential enzyme cofactor for cellular metabolism. Inadequate levels of thiamine have a devastating impact on actively metabolic tissues such as the brain parenchyma. Thiamine insufficiency (TI) is in fact a well-established comorbidity of age-related neurodegenerative disorders. In Alzheimer disease (AD) TI correlates with neurotoxicity, plaque formation, energy hypometabolism, inflammation and oxidative stress. Despite the well-established relationship between reduced thiamine levels and AD neuropathology, the molecular mechanism on how chronic TI promotes AD-like pathology is still lacking.
Our previous work has shown that TI stabilizes Hypoxia Inducible Factor -1α (HIF-1α), the main transcription factor involved in hypoxic stress, through an oxygen independent mechanism (pseudo-hypoxia). After cerebral ischemia, HIF-1α has been shown to potentiate the amyloidogenic cascade via direct transcriptional activation of β-secretase-1
(BACE-1). BACE-1 cleaves the Amyloid Precursor Protein (APP) into 99-residue membrane-associated fragments (C99) involved in the generation of β-amyloid peptides.
Therefore, HIF-1α may be a critical link between thiamine insufficiency and AD pathophysiology.

METHODS

Mouse hippocampal neuronal cells (HT22) were grown in customized thiamine deficient RPMI. Cells were seeded at 10,000 cell/cm2 and treated with 10 uM of Pyrithiamine Hydrobromide (PT), 10 uM of YC1, 2 mM of octyl-a-ketoglutarate (KG) or 150 uM Dimethyloxallyl Glycine (DMOG) for 72 hours. Cells were harvested as whole cell lysates for Western blot analysis (50 ug), or RNA was isolated for Real Time PCR (1 ug). BACE-1 activity in HT22 was measured using the fluorometric BACE-1 activity kit (Biovision) following the manufacture protocol.
For transfection, HT22 were seeded at 40,000 cell/ cm2 and transfected with 2.5 ug of Dominant Negative (DN) HIF-1a or control plasmid with 0.01%
lipofectamine (Promega) in serum free RPMI medium.

RESULTS

Treatment with the thiamine antagonist Pyrithiamine activated HIF-1α and enhanced the mRNA and protein levels of BACE-1 and C99 fragments in HT22 (2-fold increase). Furthermore, a 6-fold enhancement in BACE-1 activity was also reported.

Supplementation of the Prolyl Hydroxylase (PHD) activator Octyl-α-ketoglutarate, attenuated HIF-1α activation, reduced expression and activity levels of BACE-1 back to control level. This suggests that TI-induced HIF-1α activation may occur through a pseudo-hypoxic mechanism via PHDs inhibition.

Additionally, pharmacological inhibition of HIF-1α via YC-1 or by a Dominant Negative HIF-1α construct decreased BACE-1 expression (0.5-fold) and activity (2-fold), confirming that the BACE-1 increase was HIF-1α dependent

CONCLUSIONS

Treatment with the thiamine antagonist Pyrithiamine activated HIF-1α and enhanced the mRNA and protein levels of BACE-1 and C99 fragments in HT22 (2-fold increase). Furthermore, a 6-fold enhancement in BACE-1 activity was also reported.

Supplementation of the Prolyl Hydroxylase (PHD) activator Octyl-α-ketoglutarate, attenuated HIF-1α activation, reduced expression and activity levels of BACE-1 back to control level. This suggests that TI-induced HIF-1α activation may occur through a pseudo-hypoxic mechanism via PHDs inhibition.

Additionally, pharmacological inhibition of HIF-1α via YC-1 or by a Dominant Negative HIF-1α construct decreased BACE-1 expression (0.5-fold) and activity (2-fold), confirming that the BACE-1 increase was HIF-1α dependent




Cerebral artery contractility regulation by simvastatin and rosuvastatin


Farzana Zerin

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Statins are amongst the most widely prescribed drugs in the world with a range of vascular effects that have been primarily attributed to the inhibition of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate-dependent Rho/ROCK signaling upon long-term treatment. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. Here, we examined acute vascular effects of therapeutically relevant concentrations (0.01-10nM) of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms using pressurized arterial myography, simultaneous of vessel Ca2+ fluorescence and diameter measurement, and pharmacological modulation.

METHODS

Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressure slowly increased to 60 mmHg. Simvastatin, rosuvastatin (0.01-10nM) and pharmacological modulators were applied after the development of myogenic tone at 60 mmHg and diameter changes tracked. For calcium imaging, the isolated arteries were preincubated in Fura-2AM (10µM) and 0.02% Kolliphor solution at room temperature for two hours before mounting in the perfusion chamber.

RESULTS

At 60mmHg, cerebral arteries developed ~36% myogenic tone, after which increasing concentrations of statins were applied. Our data showed that the application of 1nM rosuvastatin and simvastatin constricted cerebral arteries by ~26 µm and ~24µm, respectively, within 2-3 minutes of drug application. Such statin-induced vasoconstriction remained unaltered upon endothelium denudation (intact ~23µm vs denuded ~25µm), suggesting an endothelium-independent mechanism. Co-application of mevalonate did not alter the vasoconstriction either (control ~28µm vs mevalonate ~29 µm) , indicating that the effect is HMG-CoA reductase-independent. However, removal of extracellular Ca2+ with EGTA (~6µm) or the application of nimodipine (~5 µm), a selective blocker of smooth muscle cell voltage-gated Ca2+ channel, CaV1.2, each abolished cerebral artery vasoconstriction by statins, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Since Ca2+ entry into smooth muscle cells induces Ca2+ release from intracellular Ca2+ stores such as sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), we next examined the role of these Ca2+ release pathways. We found that co-application of ryanodine (control ~27µm vs ryanodine ~26 µm), a blocker of ryanodine receptor-mediated Ca2+ release, had no effects on statin-induced constriction. In contrast, statin-evoked vasoconstriction of cerebral arteries was significantly attenuated upon co-application of thapsigargin (control ~27µm vs thapsigargin ~12 µm), a blocker of SR/ER membrane Ca2+-ATPase pump (SERCA), highlighting the involvement of thapsigargin-sensitive Ca2+ stores in regulating [Ca2+]i and vasoconstriction. Simultaneous measurement of arterial Ca2+ fluorescence and diameter further confirmed the involvement of CaV1.2 channel in mediating Ca2+ entry and subsequent Ca2+ release, leading to cerebral artery vasoconstriction.

CONCLUSIONS

Altogether, our data suggests that smooth muscle cell CaV1.2 opening and Ca2+ influx is the primary mechanism underlying statin-induced constriction of cerebral arteries.





 

Digital Health

PulMon-C: A Real-time Monitoring Framework of Pulmonary Function


Md Saiful Islam

Department of Information Technology. Kennesaw State University

BACKGROUND This project will develop PulMon-C, a real-time monitoring framework of pulmonary function to diagnose COVID-19 patients who are being self-quarantined at home. The tool will identify anomalies in breathe rate and predict pulmonary deterioration to raise alert for immediate actions. The uniqueness of the tool is using non-invasive sensors placed under-mattress that are able to communicate data about the respiratory signal. The customer segment of PulMon-C will be the diagnosed COVID-19 patients and healthcare providers. PulMon-C will assist with the remote monitoring of COVID-19 patients as an urgent need in the USA and will bring larger impact in delivering medical care for worsening conditions of COVID-19 patients timely without overwhelming hospital systems that have otherwise limited capacity and resources. The goal of the research is to perform the needed R&D for real-time monitoring of COVID-19 patients, develop and test signal processing techniques, and preliminary evaluation through end users and healthcare professionals. METHODS PulMon-C consists of an under-mattress pressure sensor that senses and communicates data about the respiratory signal of the person lying on a bed. Using intrinsic mode functions (IMF) and principal component analysis (PCA) as signal processing techniques, PulMon-C is able to extract the respiration rate of a person without using other kinds of apparatus. The extracted signal is sent to a Cloud (PulMon-C Cloud) directly connected with the healthcare provider in real-time using a powerful stream-data database (Influx DB). The system provides real-time monitoring of the patient's respiratory rate by showing up-to-date stream data of the breath that is refreshed every five or less seconds. When the patient presents a rapid deterioration of the pulmonary function, the system triggers an alert to the healthcare provider, who can check the status in the system. The rapid deterioration is measured by the analysis of shortness of breath indicators in the time and frequency domain and the alerts are configured in a powerful visualization tool (Grafana). RESULTS With our initial prototype, we were able to collect pressure data into the cloud service. The prototype consists on a Raspberry Pi 4 that is connected to a BPM180 pressure sensor that transfers the data using a 12C Shield digitizer. The Raspberry Pi incorporates a local database that is accessed via Wireless by the Cloud that contains a powerful data visualization tool. The data is collected continuously by a service that reads the digital data and save it into the database. Because the access is done in real-time, the user can see the pressure data every second. We expect that the tool will be a unique addition for affordable care of tracking the pulmonary function and monitoring respiration rate from the comfort of their home; the device can provide real-time transferring of information to the healthcare provider too. The long-term impact includes a real-time report of the diagnosed patients by hospitals and clinics and getting alerts when a patient is in rapid pulmonary deterioration. Healthcare providers can plan in advance their available resources as PulMon-C will provide a prediction of future patient's condition (using machine learning) based on the current and past hours/days of monitoring. PulMon-C analyzes the data and provides real-time information directly to the hospital, which is convenient and useful for both the healthcare provider and the patient. This technology will have a major social impact on diagnosed individuals. CONCLUSIONS Incorporating a real-time and easy-to-use system for monitoring COVID-19 patient's pulmonary function from home is a life-changing technology for patients and healthcare providers. PulMon-C will be the first real-time monitoring framework of COVID-19 patients who are being self-quarantined from home.





 

Medical Technology and Devices

Additive Manufacturing to Create Antimicrobial Nitric Oxide Releasing Surfaces


Manjyot Kaur Chug University of Georgia BACKGROUND Each year 99,000 deaths are reported due to hospital-acquired infections. A great extent of nosocomial infections in the patients' source from medical devices that feature a suitable surface to promote biofilm formation. One approach to addressing these infections is to create bioactive devices with nitric oxide-releasing (NO) properties. Nitric oxide has been extensively utilized for treating medical device infections due to its inherent antimicrobial and anti-inflammatory properties. Polymers releasing NO at endogenous rates (ca. 0.5-4x10-10 mol cm-2 min-1) possess similar antimicrobial properties. To overcome the design of geometrically complex devices, additive manufacturing technology was utilized to print custom designed films (porous and solid) using polycarbonate-based silicone elastomer (ChronoSil). The films were impregnated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) using a solvent-swelling process to generate antibacterial interface. Based up on the modulation of structure via distinct porosities, the NO-releasing films were studied for real-time NO release, SNAP leaching and impregnation, and antibacterial properties, that are crucial to enable clinical translation. METHODS

Polycarbonate-based silicone elastomer (PCU-Sil) films were fabricated with different porosities (60% porous, 40% porous, solid, and capped) using 3D printer. Films were soaked in SNAP (100 mg/mL) dissolved in 40% methyl ethyl ketone and 60% methanol for 2 h and dried under ambient conditions for 24 h. The NO-releasing PCU-Sil were incubated in PBS-EDTA at 37°C to determine the real-time NO release under physiological conditions using Nitric Oxide Analyzer 280i (Frederick, CO). The SNAP impregnation and leaching studies for the 3D printed films were conducted using UV-Vis spectroscopy. SEM and EDS mapping were used to examine the morphology and elemental composition of the films before and after SNAP impregnation. Finally, NO releasing films were assessed for antibacterial ability using 24 h bacterial adhesion against Staphylococcus aureus strain of bacteria frequently found in medical device related infections.

RESULTS The UV-vis studies demonstrated highest amount of SNAP in porous samples (60% and 40%) followed by solid and capped. All the samples exhibited higher levels of NO release in the initial days. While the porous samples demonstrated continued NO release at relatively higher levels up to 14 days, solid and capped samples had diminished flux levels after day 5. NO releasing films exhibited > 99% suppression of S. aureus adhesion on the film surface (p < 0.05) after 24 h. CONCLUSIONS The SNAP impregnated 3D-printed PCU-Sil films provide > 7 d of NO release at physiologically relevant levels. This study highlights the importance of additive manufacturing in biotherapeutics and paves a new path for developing patient specific antimicrobial medical device with customized NO-releasing capability. The closely regulated personalized bioactive polymers can help overcome the design of geometrically complex devices, the potential toxicity to the patient from the biofilm colonization, and emergence of antimicrobial resistance.




Characterization of the Combined Therapy of a Nitric Oxide (NO) Donor Molecule and Cerium Oxide Nanoparticles for Antimicrobial Applications


Lori Estes

School of Chemical, Materials, and Biomedical Engineering, College of Engineering, University of Georgia

BACKGROUND Broad-spectrum antimicrobials are needed towards mitigating the issues of antibiotic-resistant infections. It is highly important to formulate new antimicrobials by combining agents with different mechanistic and broader microbial targets. A combined antimicrobial solution could be a highly critical step towards developing the strategy to prevent polymicrobial infection. Herein, we have investigated the interaction and antimicrobial potential of a solution that contains cerium oxide nanoparticles (CNP) and a nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP). It is hypothesized that these two agents induce synergistic effects and would provide broad antimicrobial effects since CNP is known to be an effective antifungal agent while NO released by SNAP is known to be a potent bactericidal agent. METHODS

Different concentrations of SNAP and CNP were combined in a solution and tested for colloidal stability, NO release, mammalian cell cytotoxicity, and antimicrobial efficacy against Staphylococcus aureus, Escherichia coli, and Candida albicans, accounting for Gram-positive, Gram-negative, and fungi, respectively.

RESULTS

The combination of SNAP and CNP led to decreased colloidal stability but increased biocompatibility compared to CNP and SNAP alone, respectively. The scavenging capabilities of CNP allowed for a prolonged NO release from the combination solution. SNAP and CNP combined in equimolar solution of 3 mM were found to be highly effective for all microbes tested compared to higher amounts of the treatments required individually.

CONCLUSIONS These results hold a promising outlook toward the development of broad-spectrum antimicrobial coatings and films with the potential to prevent polymicrobial infections and further enhance biomedical device usage and applications.




A Recipe for Academic Labs to Produce SARS-CoV-2 RT-qPCR Test Kits


Sara Fakhretaha-Aval

Georgia Institute of Technology

BACKGROUND The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 30 million people, claimed many lives, and substantially disrupted activities in the public and private sectors. Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. METHODS

Here we describe our in-house Master Mix and RT-qPCR assay for detection of SARS-CoV-2. We discuss preparation of singleplex and multiplex primers and probes with CDC sequences that can be used with commercial enzyme Master Mixes. In addition, we present the production of reverse transcriptase (RT), Taq DNA polymerase, and ribonuclease inhibitor (RI) proteins, and the formulation of a working 1-step enzyme Georgia Tech Master Mix (GT-Master Mix) for use with our primers and probes.

RESULTS

We formulated a functional SARSCoV-2 assay that compares favorably to commercially available RT-qPCR kits. Our assay comprises a Master Mix as well as primers and probes identical to validated CDC sequences. GT-Master Mix is composed of affinity-purified GT-rRI, GT-His-Taq, and GT-MMLV reverse transcriptase, a compatible buffer containing cationic cofactors, plus BSA and trehalose for stability and long-term storage. In GT- Master Mix, the efficiencies of our GT primer and probe sequences met (multiplex) or closely approached (singleplex) our efficiency target of 90- 110% with high linearity (r 2 >0.990), indicating minimal primer dimers or non-specific amplification. The efficiency and linearity of our multiplex kit over a five-log concentration range is competitive with other kits that have received EUA for SARS-CoV-2 testing. In sum, our GT RT-qPCR assay, composed of proteins and enzymes produced in house, with either singleplex or multiplexed primers and probes kit exhibits a high level of qPCR efficiency and storage stability.

CONCLUSIONS The goal of our project was to create contingency SARSCoV-2 diagnostic test components in the face of supply line insecurity. We translated published information about RT-qPCR and sophisticated commercial kits into a series of fundamental protocols, executable with consumables and equipment routinely used in academic biochemistry laboratories. Our published blueprint should be readily reproducible by research teams at other academic institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource limited settings. In the long term, our protocols should be adaptable to the detection of other novel or seasonal infectious viral agents.




Rapid Susceptibility Testing from Positive Blood Cultures


Alexandra Filbrun

Georgia Institute of Technology

BACKGROUND In large part due to the rapid development of antimicrobial resistance, bacterial bloodstream infections (BSIs) remain a major cause of mortality and morbidity despite the wide availability of antibiotics. With exceedingly low bacterial burdens of 1-100 colony-forming-units per mL blood, conventional diagnosis relies on lengthy blood culturing and purification steps prior to already slow identification and antibiotic susceptibility testing (AST). This >60-hour time-to-result imposed on actionable treatment determinations negatively impacts patient outcomes and increases proliferation of antimicrobial resistance through the misuse and overuse of broad-spectrum antibiotics. Consequently, the development of novel technologies capable of rapidly recovering bacteria originally present in high blood backgrounds is increasingly important. METHODS

To address this need, we developed a novel bacterial separation technology from positive blood cultures that couples selective lysis with direct, centrifugation-based bacterial recovery.

RESULTS

Evaluated against the most common BSI-causing bacteria pathogens: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria can be recovered in <15 minutes with minimal sample handling. ASTs are readily performed with excellent correspondence to much slower ASTs performed with VITEK-2 Instrumentation.

CONCLUSIONS Overall, this direct from positive blood culture AST enables susceptibility determinations to be assessed in 3-6 hours from blood culture positivity, with minimal sample handling and processing.




Functional Outcome and Self-reported Outcome Comparison Between Osseointegrated and Socket Prosthesis for Lower Extremity Amputees: A Meta Analysis


Jacob Lonowski

Georgia State University Department of Physical Therapy

BACKGROUND

Osseointegrated (OI) prostheses are becoming a more attractive option for patients with an amputation, especially for those who have had chronic skin issues and recurrent infections with socket prosthetic use. Despite the mounting number of publications which support OI prostheses, a meta-analysis of related outcomes has not yet been conducted. Thus, this study's aim is to provide a meta-analysis of self-reported and functional outcome comparisons between OI prostheses and traditional socket prostheses in lower extremity (LE) amputees. The selected functional outcomes for comparison, the 6 Minute Walk Test (6MWT) and Timed Up and Go (TUG), are staples for assessing the functionality of an individual and are valid and reliable measurements. The self-reported outcomes, the Questionnaire for Persons with a Transfemoral Amputation (QTFA) and the Short Form 36 (SF-36), are both valid and reliable measures for the assessment of this population. Both the QTFA and the SF-36 are questionnaires which assess the status of an individual across a spectrum of physical and mental qualities.

METHODS

Six databases were searched including Cochrane, Pubmed, Medline, EMBASE, CINAHL, and Web of Science. Literature search was independently conducted by two reviewers. 1434 studies met the search criteria, 277 studies screened after the removal of duplicates, 23 full text articles were reviewed, and 15 studies were eligible for final inclusion. Two reviewers assessed study quality and extracted data independently. Analysis of the extracted data was performed using 'Comprehensive Meta-Analysis.'

RESULTS

The 15 included studies compared outcomes of 6MWT, TUG, QTFA, and SF-36 between OI prostheses and socket prostheses. The analysis favored OI prostheses with large effect sizes of statistical significance for all noted outcomes; 6MWT (ES=2.576, P<0.001), TUG (ES=2.156, P<0.001), QTFA (ES=2.981, P<0.001), and SF-36 (ES=2.068, P<0.001) in LE amputees.

CONCLUSIONS OI prostheses were found to produce superior functional outcomes in both 6MWT and TUG as well as superior self-reported metrics in both QTFA and SF-36 scores in LE amputees.




A Review of Fall Detection and Fall Prediction Systems


Lorna Migiro

Kennesaw State University

BACKGROUND

Falls threaten seniors' safety, their independence and generate enormous economic and personal costs. Methodologies for anticipating falls are essential as the benefit of prevention far outweigh those of rehabilitation. There is a current surge of smart sensors and Internet of Things being incorporated in critical fall detection and prediction safety applications. However, in recent years, there is a lack of survey reviewing and discussing on these novel sensors, technologies and algorithms introduced and employed as well as the emerging challenges and new opportunities. This paper is a survey of the state of the art of technology-based fall detection and prediction systems, which are identified from a systematic review of studies presented in contemporary research literature. It aims to serve as a point of reference for future research on the mentioned systems

METHODS

The review of the current literature was performed to explore sources of literature from ACM, IEEE digital library, PubMed, web of science and science direct. We have performed searches using the keyword "fall detection", "fall prevention", "smart fall detection", "smart fall prevention". We limit the articles between year 2010 and 2020. We gave priority to papers whose intention was broad characterization of the tech - based fall detection and prediction systems

RESULTS

This review identified 41 projects that used wearable systems and 23 projects using non-wearable systems. Context aware and vision-based systems were found to be less popular with the elderly as they lack privacy. 90 % of approaches reviewed leverage on assimilating the smartphone sensor data in generating fall prediction machine learning algorithms. Multi sensor approaches that combine acceleration magnitude, sensor velocity, and body posture give the highest fall sensitivity and the lowest false positive rate

CONCLUSIONS Fall detection and prediction systems whether physiology or kinematics both rely on sensors and fall factors. With smart sensors and Internet of Things (IoT) developing rapidly, this field has made great progress. Whereas fall prediction systems are typically developed to estimate real-time or future fall risk, real time emulations among the elderly is difficult to obtain and all studies reviewed use young adults whose representation still remains controversial, thus poses challenge on how to put this studies into practice




A Portable Optoelectronic Device for Lung Disease Diagnosis Through an Exhaled Breath


Sanjay Sarma Oruganti Venkata

University of Georgia

BACKGROUND

Diagnosis of lung diseases in both adults and children begins with the physical observation of the patient's respiratory efforts and comparing them to the respiratory rate thresholds set forth by the WHO. Beyond breath rate identification, other diagnostic measures, including auscultation by stethoscope, cultures, blood serum examination, complete blood cell count, and chest radiography (x-rays). Regardless of the identification through these methods, they lack the capability of quickly and accurately identifying the infection-causing pathogen-a critical aspect to consider when deciding treatment course as there are a wide variety of treatments available.

METHODS

Considering the shortcomings in the current diagnostic procedures, we developed a highly portable online device for detecting the particle densities and pathogen composition in a breath. Our invention under progress is a highly portable optoelectronic device that detects a breath's optical scatter characteristics passed/ blown through it. To assimilate the breathing in humans, we conducted preliminary experiments by blowing aerosol particles into the device for detecting the particle density and flow characteristics.

CONCLUSIONS We further plan to conduct experiments with various bacteria types suspended in the aerosols similar to an exhaled breath. In the current poster, we introduce the device and the results obtained from our preliminary experiments.




Characterization of E-Cigarette Aerosol Deposition in the Human Lung Airways With a Comparison to Conventional Tobacco Cigarettes


Sara Spalding

School of Engineering, Mercer University

BACKGROUND

In recent years, electronic cigarettes (e-cigarettes) have been under scrutiny due to their adverse health effects, although they had been advertised as a healthier option for current smokers. The effects of e-cigarette flavors and various flow rates on aerosol particle concentrations were evaluated. Analysis of inhaled aerosol particles in human airways using analytical lung morphometry software was performed. Additionally, a comparison of aerosol characteristics of the e-cigarette and the conventional tobacco cigarette was performed.

METHODS

Aerosol characteristics from e-cigarettes and tobacco cigarettes were measured using a Wide Range Particle Spectrometer (WPS, 1 LPM), which can measure the concentration of aerosols in the range of 10 nm to 10 microns. The Multiple-Path Particle Dosimetry Model (MPPD) was used to calculate the amount of aerosol deposited in the lung airways.

RESULTS

The maximum particle number concentrations in ambient air, filtered air, and e-cigarette aerosols during the experiment were 57, 7, and 113,428 number of particles/cc, respectively. The influence of ambient and filtered air is negligible for particle concentration measurements. It was found that there is no statistical difference in the particle concentrations of different flavors and flow rates. The experimental data used in the MPPD analysis was that of the 5% nicotine, 1.7 LPM, mint e-cigarette. Within the MPPD analysis, we compared the Stochastic Model, Stochastic 25 bpm (lung disease), Stochastic 6 bpm (breathing impairment), Yeh Schum 5 Lobe, Age-Specific 14 years old, and Age-Specific 21 years old. In the pulmonary region Age 21 and Stochastic 6 bpm had higher particle deposition, Stochastic 25 bpm had lower particle deposition. In the whole lung, Age 21 and Stochastic 6 bpm models had higher particle deposition, Age 14 had lower particle deposition. It was found that 13.04% of the cigarette smoke was deposited in the lungs, of that amount 62.89% deposited in the pulmonary region. In comparison, 6.97% of E-cigarette smoke deposited in the lungs, of that amount 44.28% deposited in the pulmonary region.

CONCLUSIONS When comparing the different MPPD models, the amount of particles deposited by the e-cigarette aerosol is dependent on the age and breathing ability of the user. Young adults had higher deposition in the whole lung and the pulmonary region. In reference to the e-cigarette aerosol, the highest amount of particles were deposited in the pulmonary airways of the lungs where O2-CO2 exchange occurs. This potentially causes inflammation of the pulmonary airways, which would hinder breathing. When comparing conventional cigarette smoke to e-cigarette aerosol, the conventional cigarette smoke deposits more particles in the lungs than e-cigarettes.




Assessing Improvements in the Quality of Life of Parents of Children with Down Syndrome After Using MapHabit's Visual Mapping Software


Kaylin White

MapHabit Inc.

BACKGROUND

There is a growing body of evidence that assistive digital technology may improve the Quality of Life (QoL) of individuals with intellectual disabilities. The MapHabit System (MHS) is an example of such a technology for it provides visual-mapping, step-by-step cues containing photos and audio, to assist users in completing tasks. The MHS has previously been used amongst memory-impaired individuals and was shown to facilitate the accomplishment of their activities of daily living (ADLs) more independently and improve the overall quality of life. Given the MHS promotes independence, we decided to explore the potential benefits of the MHS for both individuals with developmental disability and their caregivers.

METHODS

To address this possibility, a focus group of 6 parents was provided the MHS's visual-mapping software to implement into the daily routines of their children with Down syndrome for a 4-week period. The children of the parents within this focus group were between the ages of 5 and 17 years old. Throughout the trial period, participants were interviewed on a weekly basis to assess progress with the MapHabit app and to assist with map building and customization. At the end of the 4-week period, the parents completed an 18 item QoL assessment and an assessment of user satisfaction.

RESULTS

Each of the parents included in the trial reported some positive change or much positive change in one or more of the in the following areas: Completed activities of daily living more quickly, increased independence, increased social interaction, decreased frustration, increased social engagement, and/or overall improved QoL. The net promotor scores, indicative of participant satisfaction, obtained showed that parents found using visual maps with their children was satisfying and that they would recommend the MHS to a friend or colleague.

CONCLUSIONS The positive findings reported in this proof-of-concept study is the first instance of the MHS being utilized by individuals with developmental disability. These initial findings demonstrate that the MHS addresses the need for a personalized low-risk, low-cost, readily available, and portable assistive technology tool that assists with independence amongst families with children with Down syndrome. Furthermore, the MHS may be applicable to a broader range of neurodiverse individuals across the lifespan.





 

Molecular and Biological Research

Activity in Rhodococcus rhodochrous after PEGylation


Palmer Amos Georgia State University BACKGROUND

Acute lymphoblastic leukemia (ALL) is a cancer with roughly 6000 new cases diagnosed annually in the US, with a higher prevalence of children (Inaba 2013, Pui 2008). In ALL bone marrow prioritizes the overproduction of incompetent lymphoblasts, disrupting the balance of blood cells, leading to lower red blood cell and platelet count. The disruption causes shortness of breath, easy bruising, and prolonged bleeding. The cancerous lymphoblasts are incapable of synthesizing the amino acid asparagine needed for cellular reproduction, and to reproduce asparagine must be taken up from the bloodstream. Aspariginase is an enzyme which converts asparagine into aspartic acid and ammonia, injections of asparagine into patients with ALL has been shown to slow progression, allowing other treatments to take effect. The current methods for obtaining asparaginase utilize gram-negative bacteria such as Escherichia coli and Erwinia chrysanthemi. The current process for obtaining asparaginase from E. coli and E. chrysanthemi. A buildup of endotoxins derived from the lipopolysaccharide (LPS) cell wall from E. coli, E. chrysanthemi and other gram-negative bacteria can contribute to septic shock. (Landesman 1978)
Rhodococcus rhodochrous (RR) DAP 96253 is a non-pathogenic gram-positive soil bacterium which exhibits asparaginase activity when induced with urea and cobalt during growth. The gram-positive bacteria possess less LPS and fewer endotoxins than gram negative bacteria. PEGylation is the process of adding a poly-ethylene glycol chain to a protein to create a hydrophobic bubble around the protein, and is widely used in pharmaceutical applications as a preservative and to prevent the immune response from prematurely dismantling the target protein.

METHODS Asparaginase purification was performed with dialysis followed by Ion-exchange chromatography. 2-picoline borane, a nontoxic reducing agent facilitates bonding via reductive amination. Using the method for detection of ammonia developed by Fawcett and Scott CONCLUSIONS We are able to show asparaginase activity after PEGylation a week after PEGylation.




Detection of SARS-CoV-2 in the Environment


Jessica Bowman

Georgia Institute of Technology

BACKGROUND

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a severe and global impact. Identification of asymptomatic yet contagious individuals is key to controlling spread of the virus; however, testing each individual with frequency is resource demanding and logistically challenging. SARS-CoV-2 can be deposited to the environment from contagious individuals through coughing, sneezing, touching and other mechanisms. The half-life of viable SARS-CoV-2 in aerosols and on common surfaces has been demonstrated sufficient to transmit the disease.

METHODS We approached the issue of environmental risk by developing methods for collection and detection of SARS-CoV-2 nucleic acids from air and surfaces. We used swabbing techniques to collect on surfaces, and are testing multiple apparatus, including filtration and liquid impingement, to collect particles from air. Once collected and treated, environmental samples were tested for SARS-CoV-2 with the same sensitive and specific RT-qPCR technique used for diagnosis of potentially infected individuals. RESULTS

We identified cheap, effective, available and reliable swabs for collection of SARS-CoV-2 nucleic acids from surfaces. We also identified a suitable medium for wetting the swab prior to sample collection, and a method for lysing the virus and protecting viral RNA from degradation that did not interfere with downstream RT-qPCR. A slight modification on the RT-qPCR technique was used to differentiate wild viral RNA from environmental contamination by SARS-CoV-2 test materials. Standard curves generated with heat-inactivated virus, or SARS-CoV-2 test materials, could be used to quantitatively estimate the amount of SARS-CoV-2 nucleic acid found in the environment. Apparatus and methods for collection and detection of virus from air with similar techniques are feasible.

CONCLUSIONS Air and surfaces can be monitored for SARS-CoV-2 nucleic acids. Though the presence of nucleic acids does not necessarily indicate infection risk, their presence indicates the presence of potentially infective particles. Environmental monitoring for SARS-CoV-2 using RT-qPCR is sensitive and quantitative, and can be used to understand and control risk in public spaces.




DNA De-methylation Early in Life Underlies Sex Differences in Estrogen Receptor Alpha Expression in Specific Hypothalamic Areas


Iagn Nino Kenji Cabahug

Georgia State University

BACKGROUND

Neural sex differences may entail differential gene expression leading to differences in cell phenotype. For example, male rodents have more calbindin-expressing neurons (CALB) in the sexually dimorphic nucleus of the preoptic area (SDN-POA) than do females, whereas females have a greater number of neurons expressing estrogen receptor alpha (ERa) in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and the arcuate nucleus (ARC). Recent studies in our lab (Mosley et al., 2017; Cisternas, Cortes et al., 2020) suggest that DNA methylation is an important mechanism underlying sexual differentiation of cell phenotype in the brain. DNA methylation is catalyzed by DNA methyl transferases, enzymes which add methyl marks onto mainly cytosine residues. Conversely, DNA de-methylation is catalyzed by ten-eleven translocation (TETs) methylcytosine dioxygenases, which remove these methyl marks through a series of steps. Expression of both enzyme groups peaks early in life in the brain (Cisternas et al., 2020), suggesting a crucial role of epigenetics in neural development. In support of this, we demonstrated that early life inhibition of DNA methylation has lasting effects on CALB and ERa expression in the POA and VMHvl, leading to reduction or elimination of sex differences in adulthood (Mosly et. al, 2019). To our knowledge, no one has tested the role of TET enzymes in establishing sex differences in the brain. We hypothesized that the removal of DNA methyl marks is an equally important process of sexual differentiation of cell phenotype and predicted that interfering with TET enzyme expression early in life would disrupt sex differences in ERa labeling.

METHODS We injected small interfering RNA (siRNA; 400-500 pmol) targeting Tet2 and Tet3 via intracerebroventricular injections into postnatal day (P) 5 male pups. We chose this age and sex because males express higher levels of Tet2 and Tet3 relative to females in the hypothalamus on P7, and it takes ~48 hours for siRNA to reduce expression. We injected non-targeting siRNA as a control in same-age male and female pups. We collected brain tissue at P25 and performed immunohistochemistry against ERa. Labeling of ERa in the ARC and VMHvl was compared across groups using ImageJ, and statistical analyses were conducted using RStudio. RESULTS

One-way ANOVA revealed a significant difference between control males, siRNA-treated males and control females in ERa labeling in the ARC (F (2,20) = 4.662, p = 0.0218). As expected, females had more ERa labeling than males, although only marginally significant (p = 0.053). ERa labeling was increased to female levels in siRNA-treated males compared to control-males, (p = 0.038). In contrast, there was no effect of siRNA treatment in the neighboring region, the VMHvl, which also has a sex difference in ERa expression.

CONCLUSIONS We found that siRNA-mediated degradation of TET mRNA through ICV treatment on P5 led to a robust down-regulation of Tet2 and Tet3 expression in the hypothalamus. Furthermore, this reduction in Tet2 and Tet3 expression had a significant effect on ERa labeling in the ARC, but not in the VMHvl. This suggest region-specific modulation of gene expression via DNA demethylation.




Perigestational Exposure to Morphine Leads to Immune Deficits in Male and Female Rats


Hannah Harder

Georgia State University

BACKGROUND

Neonatal opioid withdrawal syndrome, or NOWS, occurs when women use opioids during pregnancy, causing the fetus to become dependent on opioids. At birth, the child is removed from the opioid source and experiences withdrawal symptoms, including autonomic dysfunction, hypersensitivity, and gastrointestinal distress. Clinical chart reviews have associated NOWS with an increased risk of hospitalization with infections, and preclinical evidence suggests opioids are immunosuppressive in adult animals. Therefore, we hypothesize that perigestational opioid exposure leads to long-term immune dysfunction.

METHODS Female Sprague-Dawley rats at P60 are implanted with iPrecio® microinfusion pumps, and given 10 mg/kg morphine/day in 3 doses. The following week, animals are bred to induce pregnancy and the dose is increased to 12 mg/kg/day, and further increased weekly to 14 and 16 mg/kg/day. At E18, doses decrease from 3 to 2 times a day. At birth, dams continue to receive morphine, and pass it to their pups through milk. When the pups reach P5, the dams' morphine dose is slowly decreased, dropping to 0 mg/kg/day by P7. This model represents the clinical features of maternal drug use in humans, including pregestational, gestational, and post-partum drug use, pulsatile dosing, and dose modulation throughout pregnancy. Pups receive indirect morphine throughout gestation and early development during critical periods of brain and immune system development. As adults, morphine-exposed male and female offspring are implanted with temperature loggers and given an immune challenge of lipopolysaccharide, which induces a characteristic fever and sickness behavior response. Fever and sickness behavior is measured for six hours post-LPS injection. Sickness behavior is scored from 0 to 8, with larger numbers representing more sick animals. RESULTS

Physiological data from morphine-exposed pups shows minor differences in weight during early life (~10-20% lower body weight). Morphine-exposed males showed a hypothermic response to LPS, as opposed to the typical hyperthermic response seen in vehicle-exposed males and females. Vehicle-exposed males and females had a net temperature change over six hours of approximately 2-3 ℃, while males had a net temperature change of -1 ℃. Interestingly, morphine-exposed females showed a net temperature change of 0 ℃, suggesting an equal hypothermic and hyperthermic response. Sickness behavior was equal between morphine- and vehicle-exposed males, while morphine-exposed females show increased sickness behavior vs. vehicle-exposed females (2 vs 1 on the sickness scale).

CONCLUSIONS We conclude that morphine-exposed male and female pups show altered immune function, both fever and sickness behavior, after LPS challenge, and that these effects are sex-dependent with morphine-exposed males potentially showing a more severe deficit. Opioid exposure during gestation likely creates long-term immune deficits due to developmental changes in the immune system, suggesting that infants born with NOWS may be at risk for immunosuppression.




Empagliflozin relaxes systemic mesenteric arteries via the activation of voltage-dependent K+ (KV) channels in arterial smooth muscle cells


Ahasanul Hasan

Mercer University

BACKGROUND

Empagliflozin, a sodium glucose cotransporter-2 (SGLT2) inhibitor, belongs to a new class of antidiabetic drugs that, in addition to reducing glucose reabsorption through kidneys, produces favorable cardiovascular effects, including a reduction of blood pressure. However, it is not clear if empagliflozin directly regulates contractility of resistance mesenteric arteries. We therefore sought to investigate direct vascular effects on empagliflozin on resistance mesenteric arteries and underlying molecular mechanisms.

METHODS To examine if empagliflozin regulates arterial contractility, we performed pressurized arterial myography on freshly isolated, ex vivo preparation of resistance-size (150-300µm), mesenteric arteries from 6-9 weeks old Sprague Dawley rats. To study the role of endothelial signaling, endothelium-intact and -denuded mesenteric arteries were used. Endothelium was denuded by slow passage of air bubbles through the vessel lumen. Absence or at least 70% reduction of acetylcholine-induced vasodilation was used as the indicator of endothelium denudation. RESULTS

Our pressure myography data showed that empagliflozin produced a concentration-dependent (0.001-100µM) vasodilation of myogenic arteries (5.76%-13.30%, expressed as % of maximum passive diameter at 80mmHg) and phenylephrine (PE)-preconstricted arteries (3.69%-72.24%, expressed as % inhibition of PE-induced contraction). To understand the mechanism(s) underlying empagliflozin-induced vasodilation, we assessed the contribution of endothelium- and smooth muscle-specific vasodilatory signaling pathways. We first investigated the role of the endothelium in empagliflozin-evoked vasorelaxation by comparing responses of endothelium-intact- and -denuded mesenteric arteries. Our data demonstrate that the magnitude of empagliflozin-induced vasodilation was similar in endothelium-intact (51.91±1.87%) and -denuded (48.81±1.87%) arteries. We further examined the role of nitric oxide (NO)-guanylyl cyclase (GC)-protein kinase G (PKG) signaling axis. Consistently, pharmacological inhibition of eNOS with L-NAME did not alter empagliflozin-evoked vasodilation (96 ± 2.62% vs 89.5 ± 6.91%). Inhibition of GC with ODQ (34.75 ± 7.90% vs 35.43 ± 4.39%) or the inhibition of PKG with KT5823 (46.53±4.23% vs 49.70 ± 8.06%) had no significant effects on empagliflozin-induced vasodilation. We next examined the role of smooth muscle cell K+ channels that control membrane potential and arterial contractility. We found that the inhibition of KV channels with 4-aminopyridine significantly reduced empagliflozin-induced vasodilation (80.37±7.47% vs 24.92±2.92%). However, inhibition of BKCa channels with paxilline, inhibition of KATP channels with glibenclamide or the inhibition of cyclooxygenase enzyme with indomethacin were without significant effects. These data suggest that smooth muscle cell KV channels are essential for mediating vasodilatory effects of empagliflozin.

CONCLUSIONS Overall, our data suggest that empagliflozin produces mesenteric artery vasodilation via the activation smooth muscle cells KV channels, without the involvement of endothelial signaling. Future studies will investigate if empagliflozin-induced arterial vasodilation can lower systemic blood pressure in healthy and in diabetic animal models.




Diverse Roles of G-Protein-Coupled Receptor 40 (GPR40/FFA1) and GPR120 (FFA4) in the Regulation of Cell Growth and Motile Activities in Renal Cell Carcinoma (RCC)


Priyanka Florina Karmokar

Mercer University

BACKGROUND

Renal cell carcinoma (RCC) is the 16th most common cancer worldwide with roughly 400,000 new cases diagnosed annually. Even though the 5-year survival rate for early-stage RCC is 80%-90%, it is only 5% for the patients with metastasis. Unfortunately, anticancer drugs that are used to treat RCC are proven to be useful for only a small number of patients. Therefore, it is crucial to discover the novel molecular pathways underlying RCC to develop better therapies. The G-protein-coupled receptors GPR40 and GPR120, also referred to as free fatty acid receptor 1 (FFA1) and 4 (FFA4), respectively, are activated by medium- to long-chain free fatty acids. Emerging evidence suggests that GPR40 and GPR120 are implicated in the proliferation, invasiveness, and apoptosis of various tumor cells, however, the precise mechanisms of GPCRs signaling events leading to the modifications in cancer cell growth and migration are still scarce, and the roles of these receptors in RCC are unstudied to date. Here, for the first time, we reveal the effects of GPR40 and GPR120 agonism on cell proliferation and migration in metastatic tumor-derived renal cancer cells.

METHODS Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to confirm the presence of GPR40 and GPR120 transcripts. Cell proliferation was assessed by plating 50,000 cells per well, followed by treatment with GPR40 and GPR120 stimulants as noted for 6 days. Cells were counted every 24 hours. For wound-healing assay, 100,000 cells were plated on a 48-well plate and a scratch made through the center. The time to wound healing was assessed at 24, 36, and 48 hr. For migration assays, cells were plated in the top chamber of a transwell plate and cells migrated was counted by crystal violet staining after 16 hr. RESULTS

Using selective GPR40 and GPR120 agonists and antagonists, our results demonstrate that GPR40 enhanced and GPR120 suppressed cell proliferation, suggesting that the opposite effects of GPR120 and GPR40 are involved in the cell growth activity. The wound-healing assay revealed that GPR120 agonism markedly covered the wounded area compared to vehicle-controls, an effect that is blocked by a selective GPR120 antagonist. However, GPR40 agonism demonstrates no significant effect, suggesting that GPR40 may not contribute to the whole-cell mass migration. In transwell migration assay, both GPR40 and GPR120 agonists elevated the ability of single cells to respond to the chemoattractant compared to vehicle-control, an effect reversed by potent GPR40 and GPR120 antagonists, respectively; further confirming their involvement in the acquisition of migratory properties in RCC cells.

CONCLUSIONS Our present results suggest that GPR40/120-mediated signaling may be involved in the regulation of renal cancer tumorigenesis and progression on the cellular level, and mechanistic studies are underway to assess the cellular cascades that are influenced by GPR40 and GPR120.




Auxin stimulates vasodilation of resistance mesenteric arteries


Sreelakshmi Menon

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Accumulating evidence suggests that short-chain fatty acids synthesized in the human body by the action of gut microbiota on the dietary fibers by anaerobic fermentation have beneficial effects on the cardiovascular system. Here, we examined the effects of auxin, a short-chain fatty acid and a plant growth hormone found in vegetable stem tips, on the contractility of Sprague Dawlay rat mesenteric arteries using pressurized artery myography and tissue nitric oxide (NO) measurement.

METHODS Fresh isolated fourth order rat mesenteric arteries were cannulated and maintained in a temperature-controlled perfusion chamber. Arterial segments were subjected to a stepwise increase in intraluminal pressure to 80 mmHg. Development of sustained myogenic tone at 80 mmHg generated a baseline diameter after which increasing concentrations (1nM to 1mM) of auxin were applied and diameter changes tracked. Quantification of tissue NO was performed after treating the tissue with auxin. RESULTS

Our pressurized artery myography data showed that auxin produced a concentration-dependent vasodilation of mesenteric arteries, with a maximum dilation of 27 µm at 1 mM concentration. To understand the mechanism underlying auxin-induced vasodilation, we next investigated the role of endothelial NO production. Our data showed that co-application of 10 µM L-NNA, a selective inhibitor of endothelial nitric oxide synthase (eNOS), caused 100% reversal of 1 mM auxin-evoked vasodilation of mesenteric arteries. Consistent with our pressure myography data, quantification of tissue NO also demonstrated that the treatment of mesenteric arteries with auxin indeed enhanced NO production.

CONCLUSIONS

Altogether, our data unveils a novel role for auxin in producing systemic mesenteric artery vasodilation via endothelial NO production. Future studies will investigate the detailed mechanism for auxin-mediated mesenteric artery vasodilation and its relevance for systemic blood pressure regulation.




Microelectrode Sensors for MicroRNAs and COVID-19 Viral RNAs


Jonathan Padelford

Georgia State University

BACKGROUND

Rapid and accurate detection of RNA species such as microRNAs and viral RNAs is increasing essential especially with the COVID-19 pandemic due to the SARS-CoV2 coronavirus. Current fast testing tools suffer significantly from the high false negative/positive rates. Laboratory methods such as real-time polymerase chain reaction (RT-PCR) are time and resource extensive, and the analysis accuracy heavily rely on trained personnel and experience due to their multistep sample treatments. Our one-step label-free electrochemical technique developed for the quantitation of microRNAs is being redesigned for the analysis of SARS-CoV2 viral RNAs and is both highly sensitive and selective.

METHODS Gold microdisk electrodes are fabricated via benchtop methods. The radii of the Au microelectrodes are in the range of several microns, sub-micron and tens of microns. A multi-segment recognition probe, which includes a nucleic acid sequence that perfectly complement the target RNAs, will be covalently attached to the Au microelectrodes via Au-thiol bonding. Several complementary sequences to the viral RNA sequences used in RT-PCR for the detection of SARS-CoV2 are chosen given their comparable length to microRNAs (and thus affinity). The tool development and target quantitation are achieved with an array of electroanalytical methods notably cyclic voltammetry (CV) and square wave voltammetry (SWV). RESULTS

This approach is supported by our previous successes to develop sensors for the microRNA miR-122. A lower limit of detection at 0.1 femtomolar (fM) and the dynamic range up to 0.5 nanomolar (nM) has been demonstrated with the introduced signal amplification mechanism and a pristine negligible background. Importantly, the detection is one-step and completes within minutes allowing for not only an extremely low level of detection but also a quick turnaround time. The quantification in absolute abundance of the target RNAs can be achieved using the electrochemical readout which is significant for both biomedical research as well as point-of-care usage. The sensors also showed superior specificity in the discrimination against analogs such as two-base mismatches of miR-122.

CONCLUSIONS This presentation explains the design principles of signal-on electrochemical sensors for 20-30 base RNAs, for example microRNAs and the viral RNAs demonstrated to be effective in RT-PCR analysis. The one-step detection of sub-femto-molar target (microRNA-122) within minutes has been achieved which is adequate for the microRNA analysis under physiological relevant range. The versatile sensor design make it readily repurposed to target other microRNAs or viral RNAs. Additional DNA-based probes targeting COVID-19 primers have been designed and sensor development is underway. Ultimately, our goal is to offer a capable tool to alleviate the immediate testing problems related to COVID-19 and be prepared for the future (mutations or other RNA viruses).




Regulation of Arterial Contractility by Neflamapimod (VX-745), an investigational drug for Alzheimer's disease


Ajay Pandey

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Alzheimer's disease is the 6th leading cause of death in the United States affecting approximately 5 million people. Preclinical studies and data from a recent phase II clinical trial suggest that neflamapimod (previously code named VX-745), a p38α MAP kinase inhibitor, reduces inflammation and reverses cognitive deficits in Alzheimer's disease. Since vascular dysfunction is linked to neurodegeneration and cognitive dysfunction in Alzheimer's disease, candidate drugs that have inherent vasodilatory effects are likely to have positive impacts on the disease outcome. Therefore, we sought to investigate the direct vascular effects of neflamapimod and characterize underlying mechanisms.

METHODS Using pressurized arterial myography and calcium imaging, we sought to investigate the direct vascular effects of therapeutic concentrations of neflamapimod on resistance mesenteric and cerebral arteries from Sprague Dawley rats. Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressured gradually increased to 80 mmHg. After sustained development myogenic tone at 80 mmHg, neflamapimod(100pM to 10μM) and pharmacological modulators were applied, and diameter changes tracked. For calcium imaging, the isolated arteries were preincubated in Fura-2AM(10µM) and 0.02% Kolliphor solution at room temperature for two hours before being mounted in the perfusion chamber. RESULTS

Our pressure myography data showed that neflamapimod produced a dose-dependent vasodilation of mesenteric arteries, with 1 and 10μM concentrations producing ~16μm and ~37μm dilation, respectively. Such vasodilation was also seen in cerebral arteries, however, to a lesser extent (~11μm and ~17μm dilation by 1 and 10μM concentrations). To delineate the mechanism underlying neflamapimod-induced vasodilation, we assessed the contribution of endothelium- and smooth muscle-specific vasodilatory signaling pathways. The vasodilatory responses produced by the highest concentration in endothelium-intact and -denuded arteries were ~37μm and ~39μm, respectively, suggesting that the mechanism is endothelium-independent. Consistently, inhibition of endothelium-derived vasodilatory pathways, such as those mediated by endothelial nitric oxide synthase (eNOS) and PGI2 had no effect on the vasodilation either. We next examined the role of smooth muscle cell K+ channels. Our data demonstrated that neflamapimod-evoked vasodilation of resistance mesenteric arteries was unaltered by the inhibition of smooth muscle cell K+ channels such as large-conductance Ca2+-activated potassium channels (BKCa: ~36μm versus ~37μm), ATP-sensitive potassium channels (KATP: ~53μm versus ~37μm), and voltage-gated potassium channels (KV: ~28μm versus ~37μm). Overall, our data suggest that neflamapimod produces vasodilation via a smooth muscle-specific mechanism that remains to be determined.

CONCLUSIONS We found that neflamapimod produces a dose-dependent vasodilation of both mesenteric and cerebral arteries that does not depend on endothelium-derived vasodilatory mechanisms or smooth muscle potassium channels. Future studies will examine the role of PKA, PKC, Rho-kinase and MAP kinase signaling in mediating neflamapimod-evoked vasodilation of cerebral and mesenteric arteries.




Polarimetric Second Harmonic Generation can be Performed in Full-thickness Bone and is Useful in Discriminating Between Healthy and Diseased States


Emily Pendleton

University of Georgia

BACKGROUND

Bone is a unique biological composite material made up of a highly structured collagen mesh matrix and mineral deposits. Although mineral provides stiffness, collagen's secondary organization provides a critical role in bone elasticity. Together, these biological components work together to form healthy bone and a disruption in one of these elements causes deleterious effects to the formation of the other. Yet, we currently only image and assess bone health with bone mineral density. Here, we work to develop a method to analyze collagen structure in full-thickness bone and implement this method to distinguish healthy bone from diseased bone.

METHODS

Our optical setup includes a Calmar fiber laser producing 1550 nm 370 fs pulses of 3uJ energy with power modulated with a half wave-plate. The polarization angle was rotated using another half wave-plate and second harmonic signal of collagen was collected. Using established theories, we determined the angle of the collagen structures within each image. To develop this method, we used three adult mouse skulls. To analyze the difference of the collagen structure, we used 13 healthy mice and 13 mice afflicted with Hypophosphatasia (HPP), a metabolic bone disorder which inhibits mineralization. To evaluate these images, we established a novel method of integrating polarimetric images with gray-level co-occurrence matrix (GLCM) texture analysis.

RESULTS

We performed polarimetric analysis of bone collagen fibers in adult mouse skulls using second harmonic generation (SHG) imaging to evaluate lamella sheets and collagen fiber integrity in intact cranial bone. Our polarimetric data was fitted to a model accounting for diattenuation, polarization cross-talk, and birefringence. We compared our data to the fitted model and found no significant difference between our polarimetric observation and the representation of these scattering properties up to 70 µm deep. We also observed a loss of resolution as we imaged up to 70 µm deep into bone but a conservation of polarimetric response.

Once this method was established, we used it to describe collagen fiber and lamella sheet orientation as well as anisotropic values in both healthy and HPP mice. We developed a novel method of angular GLCM analysis and were able to separate these two groups with PLS-DA analysis with 0.00% misclassification rate using the leave-one-out method.

CONCLUSIONS We established the analytical use of polarimetric SHG which allows for the discrimination of collagen lamellar sheet structures in intact bone. We then used this work to describe collagen defects for the first time in HPP. Furthermore, we developed a novel angular GLCM technique that effectively separated healthy from diseased bone. Our work could allow for label-free identification of disease states and monitor the efficacy of therapies for bone disorders.




Community-level Analyses of Anthropogenically Afflicted Jamaica Bay, NY Using the 16S rRNA Biomarker


Fortino Pineda Veloz

Georgia State University

BACKGROUND

Jamaica Bay, adjacent to New York City (NYC), has been afflicted by nitrogen saturation due to urban runoff and an outdated combined sewer system; in addition to physical alterations from urban development and demand. This has resulted in recurring harmful algal blooms (HABs) and an increase in average low tide depth with 85% of the wetland area lost in the past 150 years. In the past decade, the NYC Department of Environmental Protection has allotted $520 million dollars to mitigate anthropogenic effects on the Jamaica Bay estuary and restore this harbor for flora and fauna. This study describes the microbial community structure from one-liter surface water samples taken in 2016 and implements functional prediction to ascertain activity in eutrophic waters

METHODS

The Citizens Water Quality Testing (CWQT) group collected one-liter surface grab samples at three distinct locations: Canarsie Pier (high human activity), Floyd Bennett Field (low human activity), and Paedergat Basin (Combined Sewer Overflow site). Samples had DNA extracted and went through the Illumina MiSeq sequencing by synthesis protocol to generate forward single end reads of the V3/V4 region of the 16S rRNA gene.
Downstream analysis consisted of the Quantitative Insights Into Microbial Ecology version 2 (QIIME2) using amplicon correction and quality filtering with the Divisive Amplicon Denoising Algorithm 2 (DADA2). 94,000 filtered sequences from each sample were taxonomically classified using the SILVA 128 rRNA database comprising of environmental samples. The diversity and richness within the samples were adequately represented in the Shannon index and Faith PD Plot. Sampling date and temperature were used to generate a Principle Coordinates Plot (PCoA) to visualize phylogenetic differences in cluster. The statistical relevance of each factor was quantified with p-values using a PERMANOVA analysis with 999 permutations.
The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) functional abundance prediction pipeline employed the DADA2 16S rRNA gene marker data alongside feature counts to construct a quantitative scope of gene families within the samples. Differences in functional profiles were predicted and recorded.

RESULTS

The PERMANOVA analysis found no correlation between sampling site with a p-value of 0.989, while there was significant correlation when sorting by sampling date with a p-value of 0.008 (p < 0.01). The climate over 6 days of each sampling date was investigated to create the following chart.

CONCLUSIONS

The most consist temperature over a six-day period was on August 18th, 2016 which showed congruence to the PCoA plot clustering. In addition, the most varied period was that of July 28th, 2016 which reflect the spread of its respective data points in the PCoA plot. These relations were also seen in the taxonomic profiling of each sample with a highly consistent profile belong to those samples taken on August 18th, 2016 as well as the inverse in the case of samples taken on July 28th, 2016.
Cyanobacteria dominate the community with values ranging from 30% to 40%. The secondmost dominant taxa was Proteobacteria, led by the subgroup Alphaproteobactertia then Gammaproteobacteria. The latter has been seen to be associated with shifts in water temperature within Cheasapeake Bay. The relation between these subgroups of Proteobacteria and temperature may be confounded by a synergistic companinionship with Cyanobacteria. The former degrades Dissolved Organic Nitrogen (DON) in the form of nitrates to produce Dissolved Inorganic Nitrogen (DIN), fostering phytoplankton booms. The many years of nitrogen-rich effluent have maintained this relationship and created a loss of microbial diversity. Furthermore, the microbial community is affected as the termination of phytoplankton blooms create anoxic regions in the bay via respiration of sinking biomass. Some bacteriaplankton also possess the ability to cycle carbon using nitrate in anoxic region-leading to the recurrence of blooms. This was also respresented functionally with support in photosynthetic genes.
Temperature increases the kinetics of reactions and creates a bias in metabolic activity. A more all encompassing chart is in design and will be ready. It will include all sampling organized by date rather than the metagenomic data created in KRAKEN as seen above.
In conclusion, the physical properties of Jamaica Bay and its community structure do not align well with the diversity-stability hypothesis. This hypothesis states that a diverse community is better suited to withstand destabilizing events. However, Jamaica Bay no longer reflects other estuaries diversity due to its prolonged infliction with nitrogen-loading, leading to a community built on a nutritive bias. As climate change continues and sea levels rise, one cannot be sure of the progress of restoration nor of the half billion dollar NYC initiative without increased sampling efforts. In this unique complex estuarine system, additional data must be generated to statistically correlate temporal changes within the bay's ecosystem to environmental factors in the form of a longitudinal study with autonomous abiotic sampling.




Molecular Identification and Characterization of Nitrile Hydratase in Rhodococcus rhodochrous DAP 96253


Fortino Pineda Veloz

Georgia State University

BACKGROUND

Nitrile hydratase (NHase) is an enzyme that hydrolyzes nitrile moieties into their corresponding amide form. When NHase catalyzes a reaction with the substrate acrylonitrile, acrylamide is formed. Acrylamide is the building block of polyacrylamide (PAM); a polymer used for coagulation during water treatment, gel electrophoresis chambers, and various manufactured polymers. The interest in this strain's enzymatic capability stems from the industrial leader in biotransformation--Rhodococcus rhodochrous J1. R. rhodochrous J1 is used to produce over 30,000 tons of acrylamide annually. It possesses high caliber enzymatic ability with percent yields hovering at 99% whilst bypassing acrylic acid as an unwanted byproduct.

R. rhodochrous DAP 96253 cells have been induced with urea and cobalt to increase nitrile hydratase activity which benefits the industrial upside of this strain. Low molecular weight nitrile hydratase (L-NHase) catalyzes the reaction first. Upon sensing the amide reaction product, high molecular weight nitrile hydratase (H-NHase) is induced, increasing catalytic activity of acrylonitrile. By gaining knowledge of transcriptional regulation, NHase production has the potential to be upregulated beyond its current level. In addition, understanding the genetic construct of R. rhodochrous DAP 96253 is paramount to further molecular and in-silico studies on the enzyme of interest.

METHODS

Sterile Eppendorf tubes containing 50 mg of glucose-urea-cobalt induced R. rhodochrous DAP 96253 cell paste were refrigerated for 45 minutes at -20°C. The paste was suspended in 250 μL of lysozyme (10 mg/mL) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 20 μL of mutanolysin (1 mg/mL). The mixture was incubated for an hour at 37°C with each tube gently shook every ten minutes. Following incubation, 50 μL of 0.5 M EDTA, 4M NaCl, and 10% SDS were added. Proteinase K (20 mg/mL) was added in the volume of 10 μL to each tube then incubated at 37°C for one hour. 350 μL of this mixture were extracted and mixed with one volume of phenol:chloroform:isoamyl alcohol (25:24:1). The tubes were then centrifuged at 13,000 x g for five minutes. The top aqueous layer had 300 microliters extracted and underwent liquid to liquid extraction with a half volume of chloroform:isoamyl alcohol. The solution was inverted several times and centrifuged at 13,000 x g for five minutes. The aqueous layer was again extracted, and precipitation followed with one volume of chilled isopropanol. The sample spun at 13,000 x g for 15 minutes. The resulting pellet was washed with 200 μL of 70% ethanol, centrifuged at 13,000 x g for 10 minutes, and resuspended in 100 μL of TE buffer. Quantification of yield and purity were conducted using a Thermo Scientific Spectrophotometer Nanodrop 2000C.

Genomic DNA of R.rhodococcus DAP 96253 was sequenced via the Ion Torrent Xpress Personal Genome machine through reconstruction of fragmented DNA. The outputted file contained possible protein products that were compared to the NCBI Basic Local Alignment Search Tool (BLAST) for protein sequences. The order and location (positive or negative strand) of those proteins identical or similar to NHase genes of the Rhodococcus genus were organized into Figure 2. Nucleotide sequences from the Ion Xpress output file were used to construct forward and reverse primers for future PCR. Primers will contain restriction enzyme recognition sites to ligate the nucleotide splice into a plasmid vector under a lactose operon for characterization of the expressed protein with subsequent assays.

CONCLUSIONS

A genetic framework for nitrile hydratase been established for the first time with R. rhodochrous DAP 96253. Future molecular work will confirm nitrile hydratase through ligation of this genetic splice into a plasmid vector. The vector will allow for control of expression under a lactose operon. Verification of analogous transcription factors of the DAP 96253 strain to the J1 strain will be done via knock out studies. Interestingly, there was no discovery of nnhE in R. rhodochrous DAP 96253 when analyzing the fragmented genomic library. This protein has no known function and it will be imperative to investigate if this protein is present in an alternate sequencing method as well as its role in H-NHase.




Inclusion of Ischemic Stroke Lesion Topology in a Translational Porcine Model Leads to Better Prediction of Functional Outcomes


Kelly Scheulin

University of Georgia

BACKGROUND

Predicting stroke patient functional outcomes based on acute magnetic resonance imaging (MRI) information has significant clinical value in determining prognosis and potential course of treatment. The inclusion of stroke lesion location when predicting functional outcomes is likely to improve prognostic ability as there is a direct relationship between brain structures and function. However, stroke location is under assessed in predicting functional outcomes with traditional metrics such as lesion volumes and midline shifts being more heavily used pre-clinically and clinically.

METHODS

The objective of this current study is to identify lesioned brain structures by MRI and correlate structural alterations with changes in motor function and behavior in a preclinical porcine model. Pigs (n=7) underwent stroke surgery where the middle cerebral artery was permanently occluded (MCAO). MRI was performed at 1 day (d) post-MCAO and functional gait analysis and open field testing were performed on 0d, 2d, 8d, 15d, and 27d post-MCAO. A porcine MRI brain atlas was registered to identify stroke lesion location, and linear regressions between infarcted brain structures and functional data were completed to evaluate the predictive capacity of individual brain structure lesion on neurological outcome.

RESULTS

MCAO resulted in prominent lesion volumes and decreased white matter integrity. Highly lesioned brain structures included the insular cortex, somatosensory cortices, visual cortices, temporal gyri, and putamen. MCAO severely impaired translational gait parameters, decreased voluntary movement in open field testing, and resulted in increased modified Rankin Scale (mRS) scoring. Linear regression analysis determined that lesions in the secondary visual cortex, claustrum, amygdala, and superior temporal gyrus were highly prognostic of overall gait and behavioral outcomes.

CONCLUSIONS

This regression analysis approach identified neuroanatomical structures that were predictive of stroke outcome and identified potential target structures for therapeutic intervention to facilitate optimal functional recovery.




Differential Modulation of β2AR Mediated cAMP/PKA/CREB Signaling Cascade in Normal versus Asthmatic Airway Epithelial Cells


Kirti Singh

Graduate Student, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University

BACKGROUND

Nearly 25 million people in the U.S. suffer from asthma. While there is no panacea for asthma, symptomatic relief is achieved by the current therapeutic interventions, including β2-adrenergic receptor (β2AR) agonists. Chronic usage of β2AR agonists can have serious implications like airway hyper-responsiveness and receptor desensitization, and along with unknown mechanisms, can ultimately culminate into tachyphylaxis to the response. Previously, our laboratory has extensively explored the critical role of reactive oxygen species (ROS) in modulating β2AR function. To begin to assess the physiological role of the β2AR-ROS linkage in asthma, here, we assessed Isoproterenol (ISO)-induced β2AR mediated Gαs/cAMP/CREB pathway in small airway epithelial cells from asthma patients (SAEC-A) compared to those from non-diseased control patients (SAEC-N). Given the importance of β2AR mediated cAMP production and its role in airway bronchodilation, as well as tachyphylaxis that occurs upon chronic use of β2AR agonists, our results may shed light on the role of oxidants in altering β2AR responses in asthma.

METHODS

To assess cAMP levels, cells were analyzed using highly sensitive luciferase-based biosensor. Briefly, SAEC were transfected with GloSensorTM -22F cAMP plasmid (8µg) using Lipofectamine3000. Luminescence was measured following treated with the ISO. To assess the effects of downstream cAMP-mediated transcription, cells were stimulated with H2O, Forskolin (FSK) (10 µM) & ISO (100µM) for 20 min and lysed. Lysates were resolved using SDS-PAGE and immunoprobed for phospho-cAMP response element-binding protein (pCREB), total CREB and β-actin. Similarly, SAEC-N/A lysates were assessed for Adenylyl Cyclase (AC-5/6) and Phosphodiesterase (PDE4) by immunoblot.

RESULTS

(1) β2AR agonism with ISO significantly increased cAMP production in SAEC from asthma patients compared to non-diseased control. (2) In spite of elevated cAMP production in asthmatic SAEC, both ISO and FSK were able to induce significant increase in CREB phosphorylation, leading to heightened pCREB/CREB response in SAEC-N as compared to SAEC-A. (3) ISO-mediated increased cAMP response in asthmatic SAEC compared to non-diseased control, were linked with physiologically altered expression of key cAMP mediators, specifically, heightened basal expression of AC-5/6 and PDE4, in asthmatic SAEC compared to SAEC-N.

CONCLUSIONS

ISO mediated increased cAMP production in asthmatic SAEC indicated altered agonist modulated β2AR downstream signaling in diseased versus non-diseased SAEC. Further, heightened basal expression of AC-5/6 in asthmatic SAEC supports the above mentioned consequence. In addition, elevated CREB phosphorylation in non-diseased SAEC suggests alterations of β2AR-mediated PKA/CREB signaling in asthma. Future results will probe the effects of elevated ROS in these signal cascades.




Hypoxia-Inducible Factor 1-α (HIF-1α) Regulates Alzheimer's Disease-Like Neuropathology during Thiamine Insufficiency


Maria Valle

University of Georgia

BACKGROUND

Thiamine is an essential enzyme cofactor for cellular metabolism. Inadequate levels of thiamine have a devastating impact on actively metabolic tissues such as the brain parenchyma. Thiamine insufficiency (TI) is in fact a well-established comorbidity of age-related neurodegenerative disorders. In Alzheimer disease (AD) TI correlates with neurotoxicity, plaque formation, energy hypometabolism, inflammation and oxidative stress. Despite the well-established relationship between reduced thiamine levels and AD neuropathology, the molecular mechanism on how chronic TI promotes AD-like pathology is still lacking.
Our previous work has shown that TI stabilizes Hypoxia Inducible Factor -1α (HIF-1α), the main transcription factor involved in hypoxic stress, through an oxygen independent mechanism (pseudo-hypoxia). After cerebral ischemia, HIF-1α has been shown to potentiate the amyloidogenic cascade via direct transcriptional activation of β-secretase-1
(BACE-1). BACE-1 cleaves the Amyloid Precursor Protein (APP) into 99-residue membrane-associated fragments (C99) involved in the generation of β-amyloid peptides.
Therefore, HIF-1α may be a critical link between thiamine insufficiency and AD pathophysiology.

METHODS

Mouse hippocampal neuronal cells (HT22) were grown in customized thiamine deficient RPMI. Cells were seeded at 10,000 cell/cm2 and treated with 10 uM of Pyrithiamine Hydrobromide (PT), 10 uM of YC1, 2 mM of octyl-a-ketoglutarate (KG) or 150 uM Dimethyloxallyl Glycine (DMOG) for 72 hours. Cells were harvested as whole cell lysates for Western blot analysis (50 ug), or RNA was isolated for Real Time PCR (1 ug). BACE-1 activity in HT22 was measured using the fluorometric BACE-1 activity kit (Biovision) following the manufacture protocol.
For transfection, HT22 were seeded at 40,000 cell/ cm2 and transfected with 2.5 ug of Dominant Negative (DN) HIF-1a or control plasmid with 0.01%
lipofectamine (Promega) in serum free RPMI medium.

RESULTS

Treatment with the thiamine antagonist Pyrithiamine activated HIF-1α and enhanced the mRNA and protein levels of BACE-1 and C99 fragments in HT22 (2-fold increase). Furthermore, a 6-fold enhancement in BACE-1 activity was also reported.

Supplementation of the Prolyl Hydroxylase (PHD) activator Octyl-α-ketoglutarate, attenuated HIF-1α activation, reduced expression and activity levels of BACE-1 back to control level. This suggests that TI-induced HIF-1α activation may occur through a pseudo-hypoxic mechanism via PHDs inhibition.

Additionally, pharmacological inhibition of HIF-1α via YC-1 or by a Dominant Negative HIF-1α construct decreased BACE-1 expression (0.5-fold) and activity (2-fold), confirming that the BACE-1 increase was HIF-1α dependent

CONCLUSIONS

Treatment with the thiamine antagonist Pyrithiamine activated HIF-1α and enhanced the mRNA and protein levels of BACE-1 and C99 fragments in HT22 (2-fold increase). Furthermore, a 6-fold enhancement in BACE-1 activity was also reported.

Supplementation of the Prolyl Hydroxylase (PHD) activator Octyl-α-ketoglutarate, attenuated HIF-1α activation, reduced expression and activity levels of BACE-1 back to control level. This suggests that TI-induced HIF-1α activation may occur through a pseudo-hypoxic mechanism via PHDs inhibition.

Additionally, pharmacological inhibition of HIF-1α via YC-1 or by a Dominant Negative HIF-1α construct decreased BACE-1 expression (0.5-fold) and activity (2-fold), confirming that the BACE-1 increase was HIF-1α dependent




Cerebral artery contractility regulation by simvastatin and rosuvastatin


Farzana Zerin

Department of Pharmaceutical Sciences, Mercer University

BACKGROUND

Statins are amongst the most widely prescribed drugs in the world with a range of vascular effects that have been primarily attributed to the inhibition of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate-dependent Rho/ROCK signaling upon long-term treatment. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. Here, we examined acute vascular effects of therapeutically relevant concentrations (0.01-10nM) of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms using pressurized arterial myography, simultaneous of vessel Ca2+ fluorescence and diameter measurement, and pharmacological modulation.

METHODS

Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressure slowly increased to 60 mmHg. Simvastatin, rosuvastatin (0.01-10nM) and pharmacological modulators were applied after the development of myogenic tone at 60 mmHg and diameter changes tracked. For calcium imaging, the isolated arteries were preincubated in Fura-2AM (10µM) and 0.02% Kolliphor solution at room temperature for two hours before mounting in the perfusion chamber.

RESULTS

At 60mmHg, cerebral arteries developed ~36% myogenic tone, after which increasing concentrations of statins were applied. Our data showed that the application of 1nM rosuvastatin and simvastatin constricted cerebral arteries by ~26 µm and ~24µm, respectively, within 2-3 minutes of drug application. Such statin-induced vasoconstriction remained unaltered upon endothelium denudation (intact ~23µm vs denuded ~25µm), suggesting an endothelium-independent mechanism. Co-application of mevalonate did not alter the vasoconstriction either (control ~28µm vs mevalonate ~29 µm) , indicating that the effect is HMG-CoA reductase-independent. However, removal of extracellular Ca2+ with EGTA (~6µm) or the application of nimodipine (~5 µm), a selective blocker of smooth muscle cell voltage-gated Ca2+ channel, CaV1.2, each abolished cerebral artery vasoconstriction by statins, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Since Ca2+ entry into smooth muscle cells induces Ca2+ release from intracellular Ca2+ stores such as sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), we next examined the role of these Ca2+ release pathways. We found that co-application of ryanodine (control ~27µm vs ryanodine ~26 µm), a blocker of ryanodine receptor-mediated Ca2+ release, had no effects on statin-induced constriction. In contrast, statin-evoked vasoconstriction of cerebral arteries was significantly attenuated upon co-application of thapsigargin (control ~27µm vs thapsigargin ~12 µm), a blocker of SR/ER membrane Ca2+-ATPase pump (SERCA), highlighting the involvement of thapsigargin-sensitive Ca2+ stores in regulating [Ca2+]i and vasoconstriction. Simultaneous measurement of arterial Ca2+ fluorescence and diameter further confirmed the involvement of CaV1.2 channel in mediating Ca2+ entry and subsequent Ca2+ release, leading to cerebral artery vasoconstriction.

CONCLUSIONS

Altogether, our data suggests that smooth muscle cell CaV1.2 opening and Ca2+ influx is the primary mechanism underlying statin-induced constriction of cerebral arteries.





 

Nanotechnology

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





 

Regulatory Affairs

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.