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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.





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Drug Discovery & Development

Identification of early adopters for the early glycation products, a potential dietary supplement


Tai Guo University of Georgia BACKGROUND Early glycation products (EGPs) generated in the first two steps of Maillard reaction/glycation are proteins modified with reducing sugar moieties. This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. Our preclinical studies have found that EGPs derived from whey protein isolate (WPI) and glucose were anti-inflammatory and implied a nutraceutical application. METHODS To identify the customer segments, different hypotheses were put forward, and customer interviews were conducted either in person (wearing masks), by phone or virtually using Zoom meeting. RESULTS

Eight people were interviewed based on the initial hypothesis that individuals interested in dietary supplements would buy EGPs because this product might make them healthier. We asked questions such as: How do you feel about a new dietary supplement that makes your immune system healthier? The responses were mostly negative, including that there was no need for a dietary supplement as long as you ate healthy. Our preclinical studies showed that EGPs protected mice against hyperglycemia through altering immune balance; therefore, we put forward our second hypothesis that, unlike others, our product was to meet the objectives of diabetic patients. We have interviewed additional ten individuals who either have diabetes themselves or know someone having diabetes. We asked questions such as: Do you have any concerns or unmet needs that can't be solved by your doctors? Although most of the responses were not supportive of our hypothesis, one response was intriguing: Supplements that help with recovery from inflammation are hugely popular among serious athletes. Further research found that too many hard workouts can lead to too much inflammation (Pain) in athletes, which may result in poor athletic performances, a compromised immunity and missed practices or competitions. In addition, it was found that the global whey protein usage in the sports nutrition market was $1.6 billion in 2014 and expected to reach almost $2.7 billion in 2020. The North American market was $1.0 billion in 2014 and expected to reach $1.7 billion in 2020. The global whey protein usage in the pharmaceutical and clinical nutrition market generated $484.5 million in 2014 and was expected to reach $980.9 million in 2020, while the North American market was $309 million in 2014 and expected to reach $627 million in 2020. Our product EGPs can reduce inflammation in addition to supplementing nutritious proteins and energy, which would be an ideal and healthy dietary supplement for athletes.

CONCLUSIONS

We have identified potential early adopters for EGPs, which will be tested out using our new hypothesis that athletes buy the dietary supplement (EGPs) because it can reduce inflammation and promote post workout recovery (supported by NIH R41DK121553, and in part by NSF I-Corps funding).




1-Hexene Monooxygenase as a Biological Alternative to Delay Ripening in Climacteric Fruits


Marilyn Millett Georgia State University BACKGROUND Problems with food production and distribution are commonly discussed in the popular press and in academic journals. According to the US Department of Agriculture, 40% of all crops produced in the US are lost before reaching consumer markets. A significant contributor to this is the overripening of crops in transit to market. Commonly used methods to prevent this include refrigeration or harsh chemicals. One example of such a chemical is 1-methylcyclopropine (MCP) which breaks down ethylene and slows the Yang Cycle in climacteric fruits such as bananas, thus delaying their ripening. The Yang Cycle is a metabolic pathway where ethylene is produced via the fruit that results in the ripening of climacteric fruits. However, there are biological alternatives that break down ethylene and prevent fruit ripening without the use of harsh chemicals. The microorganism Rhodococcus rhodochrous DAP 96253 is known to possess the enzyme 1-Hexene Monooxygenase (1-HMO), commonly known as an Alkene Monooxygenase (AMO), which is believed to delay fruit ripening by converting the ethylene given off by the fruit into its epoxide form. METHODS A 1X mR3A media was used to generate induced cells of R. rhodochrous DAP 96253 in a 4L flask. After four days of growth at 30°C, the cells were then harvested via centrifugation into cell paste. In whole cell experiments, 3g paste was added to sealed plastic containers with four bananas in each container. Pictures were taken every four days for 14 days. The cells are lysed via sonication and the enzyme was purified via anion exchange chromatography (AEC). Purified product was then added to fresh bananas and observed for 14 days. RESULTS

Enzymatic activity was checked in whole cells using a color change reaction and read at 600 nm. After AEC, a BCA assay was performed to verify presence of protein.

CONCLUSIONS

This experiment will demonstrate that 1-HMO is extractable and purifiable from whole cells of R. rhodochrous while maintaining activity resulting in the delayed ripening of bananas.




Hygiene Status of Fresh Peach Packing Lines in Georgia


Peien Wang The University of Georgia BACKGROUND

Current Produce Safety Rules in the U.S. requires fresh produce packing equipment be clean and maintain sanitary conditions. This study surveyed the hygiene status of selected fresh peach packing lines in Georgia.

METHODS

Surface swabbing samples (n = 464) were collected from 14 selected sites on each of the 4 fresh peach packing lines at 3 times of a day, before packing in the morning (AM), at lunchtime (NOON), and the end of the day (PM), in 3 repeated visits per line during the harvest seasons of 2018 and 2019. Each swab sample was collected from a 100 cm2 area using a sterile sponge pre-moistened with Dey-Engley neutralizing broth. Collected samples were transported to our laboratory under refrigeration condition and analyzed within 16 - 24 hours. The levels of three hygiene indicators, total aerobes (TA), total yeasts and molds (YM), and total coliforms (TC), as well as the incidences of thermo-tolerant coliforms (TTC) and enterococci (EC) in collected samples were determined. Results were analyzed using the split-plot ANOVA tests fitted in a general linear regression model.

RESULTS

Counts of the three hygiene indicators in the AM samples were significantly lower (P<0.05) than those in the NOON and PM samples. The incidence of TTC increased from 13.50% at AM to 45.83% at PM, while that of EC from 6.75% to 15.11%. Higher levels of TA, YM, and TC counts and higher incidences of TTC and EC were observed on the brushes/rollers inside the washer/waxer and optical sizer compared to other sites sampled. In comparison, harvest bins were only high in TA and YM counts. Samples from manual sorting area had the highest TTC incidence among samples from all surveyed sites.

CONCLUSIONS

Results suggest that microbial build-up on fresh peach lines is time-dependent. The study pinpointed critical sanitation control points on fresh peach packing lines.




Inhibitory activity of aqueous extracts of pomegranate peels and juice powder on Salmonella enterica subspecies enterica serotype Tennessee and Enteritidis


Weifan Wu

The University of Georgia BACKGROUND

Pomegranate is a nutrient dense fruit rich in polyphenols including a high concentration of ellagitannins that have antimicrobial properties. Inedible pomegranate peel is reported to have even higher amounts of polyphenols than the edible arils, which makes pomegranate peel a promising source of natural preservatives.This study evaluated the inhibitory effect of aqueous extracts of different pomegranate products, including three dry peel powders, a whole peel, and a juice powder against two different strains of Salmonella enterica in tryptic soy broth (TSB) or phosphate buffered saline (PBS).

METHODS Salmonella cell suspension at 10^5 cfu/ml was added into either TSB or PBS containing either 9% or 24% of the extracts prepared by two different methods. Surviving Salmonella cells were enumerated after treatment for 5 h, 10 h, 24 h at 25 ℃, and reduction of Salmonella population in relation to the control was subsequently determined.

RESULTS

Results showed that the reduction of Salmonella population in TSB was significantly higher (P≤0.05) than that in PBS. Salmonella Enteritidis had a significantly lower (P≤0.05) reduction than Salmonella Tennessee in PBS and a numerically lower (P > 0.05) reduction in TSB. The extracts from the three powdered peels were significantly more effective (P≤0.05) in reducing the Salmonella population (1.67 - 2.03 Log CFU/ml) than the extract from the whole peel (1.05 Log CFU/ml) and juice powder (0.94 Log CFU/ml). Higher dose of extracts resulted in a greater reduction in Salmonella population in TSB (3.02 Log CFU/ml) or PBS (0.66 Log CFU/ml) than media with lower dose of extracts. The level of Salmonella population reduction correlated positively with the polyphenolic content (R2 0.66 - 0.99) and titratable acidity (R2 0.72 - 0.98) in the treatment systems.

CONCLUSIONS

The study suggests that the pomegranate peels have the potential to be use as preservatives in human food or animal feed to control pathogens like Salmonella.





 

Food and Nutrition

Identification of early adopters for the early glycation products, a potential dietary supplement


Tai Guo University of Georgia BACKGROUND Early glycation products (EGPs) generated in the first two steps of Maillard reaction/glycation are proteins modified with reducing sugar moieties. This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. Our preclinical studies have found that EGPs derived from whey protein isolate (WPI) and glucose were anti-inflammatory and implied a nutraceutical application. METHODS To identify the customer segments, different hypotheses were put forward, and customer interviews were conducted either in person (wearing masks), by phone or virtually using Zoom meeting. RESULTS

Eight people were interviewed based on the initial hypothesis that individuals interested in dietary supplements would buy EGPs because this product might make them healthier. We asked questions such as: How do you feel about a new dietary supplement that makes your immune system healthier? The responses were mostly negative, including that there was no need for a dietary supplement as long as you ate healthy. Our preclinical studies showed that EGPs protected mice against hyperglycemia through altering immune balance; therefore, we put forward our second hypothesis that, unlike others, our product was to meet the objectives of diabetic patients. We have interviewed additional ten individuals who either have diabetes themselves or know someone having diabetes. We asked questions such as: Do you have any concerns or unmet needs that can't be solved by your doctors? Although most of the responses were not supportive of our hypothesis, one response was intriguing: Supplements that help with recovery from inflammation are hugely popular among serious athletes. Further research found that too many hard workouts can lead to too much inflammation (Pain) in athletes, which may result in poor athletic performances, a compromised immunity and missed practices or competitions. In addition, it was found that the global whey protein usage in the sports nutrition market was $1.6 billion in 2014 and expected to reach almost $2.7 billion in 2020. The North American market was $1.0 billion in 2014 and expected to reach $1.7 billion in 2020. The global whey protein usage in the pharmaceutical and clinical nutrition market generated $484.5 million in 2014 and was expected to reach $980.9 million in 2020, while the North American market was $309 million in 2014 and expected to reach $627 million in 2020. Our product EGPs can reduce inflammation in addition to supplementing nutritious proteins and energy, which would be an ideal and healthy dietary supplement for athletes.

CONCLUSIONS

We have identified potential early adopters for EGPs, which will be tested out using our new hypothesis that athletes buy the dietary supplement (EGPs) because it can reduce inflammation and promote post workout recovery (supported by NIH R41DK121553, and in part by NSF I-Corps funding).




1-Hexene Monooxygenase as a Biological Alternative to Delay Ripening in Climacteric Fruits


Marilyn Millett Georgia State University BACKGROUND Problems with food production and distribution are commonly discussed in the popular press and in academic journals. According to the US Department of Agriculture, 40% of all crops produced in the US are lost before reaching consumer markets. A significant contributor to this is the overripening of crops in transit to market. Commonly used methods to prevent this include refrigeration or harsh chemicals. One example of such a chemical is 1-methylcyclopropine (MCP) which breaks down ethylene and slows the Yang Cycle in climacteric fruits such as bananas, thus delaying their ripening. The Yang Cycle is a metabolic pathway where ethylene is produced via the fruit that results in the ripening of climacteric fruits. However, there are biological alternatives that break down ethylene and prevent fruit ripening without the use of harsh chemicals. The microorganism Rhodococcus rhodochrous DAP 96253 is known to possess the enzyme 1-Hexene Monooxygenase (1-HMO), commonly known as an Alkene Monooxygenase (AMO), which is believed to delay fruit ripening by converting the ethylene given off by the fruit into its epoxide form. METHODS A 1X mR3A media was used to generate induced cells of R. rhodochrous DAP 96253 in a 4L flask. After four days of growth at 30°C, the cells were then harvested via centrifugation into cell paste. In whole cell experiments, 3g paste was added to sealed plastic containers with four bananas in each container. Pictures were taken every four days for 14 days. The cells are lysed via sonication and the enzyme was purified via anion exchange chromatography (AEC). Purified product was then added to fresh bananas and observed for 14 days. RESULTS

Enzymatic activity was checked in whole cells using a color change reaction and read at 600 nm. After AEC, a BCA assay was performed to verify presence of protein.

CONCLUSIONS

This experiment will demonstrate that 1-HMO is extractable and purifiable from whole cells of R. rhodochrous while maintaining activity resulting in the delayed ripening of bananas.




Hygiene Status of Fresh Peach Packing Lines in Georgia


Peien Wang The University of Georgia BACKGROUND

Current Produce Safety Rules in the U.S. requires fresh produce packing equipment be clean and maintain sanitary conditions. This study surveyed the hygiene status of selected fresh peach packing lines in Georgia.

METHODS

Surface swabbing samples (n = 464) were collected from 14 selected sites on each of the 4 fresh peach packing lines at 3 times of a day, before packing in the morning (AM), at lunchtime (NOON), and the end of the day (PM), in 3 repeated visits per line during the harvest seasons of 2018 and 2019. Each swab sample was collected from a 100 cm2 area using a sterile sponge pre-moistened with Dey-Engley neutralizing broth. Collected samples were transported to our laboratory under refrigeration condition and analyzed within 16 - 24 hours. The levels of three hygiene indicators, total aerobes (TA), total yeasts and molds (YM), and total coliforms (TC), as well as the incidences of thermo-tolerant coliforms (TTC) and enterococci (EC) in collected samples were determined. Results were analyzed using the split-plot ANOVA tests fitted in a general linear regression model.

RESULTS

Counts of the three hygiene indicators in the AM samples were significantly lower (P<0.05) than those in the NOON and PM samples. The incidence of TTC increased from 13.50% at AM to 45.83% at PM, while that of EC from 6.75% to 15.11%. Higher levels of TA, YM, and TC counts and higher incidences of TTC and EC were observed on the brushes/rollers inside the washer/waxer and optical sizer compared to other sites sampled. In comparison, harvest bins were only high in TA and YM counts. Samples from manual sorting area had the highest TTC incidence among samples from all surveyed sites.

CONCLUSIONS

Results suggest that microbial build-up on fresh peach lines is time-dependent. The study pinpointed critical sanitation control points on fresh peach packing lines.




Inhibitory activity of aqueous extracts of pomegranate peels and juice powder on Salmonella enterica subspecies enterica serotype Tennessee and Enteritidis


Weifan Wu

The University of Georgia BACKGROUND

Pomegranate is a nutrient dense fruit rich in polyphenols including a high concentration of ellagitannins that have antimicrobial properties. Inedible pomegranate peel is reported to have even higher amounts of polyphenols than the edible arils, which makes pomegranate peel a promising source of natural preservatives.This study evaluated the inhibitory effect of aqueous extracts of different pomegranate products, including three dry peel powders, a whole peel, and a juice powder against two different strains of Salmonella enterica in tryptic soy broth (TSB) or phosphate buffered saline (PBS).

METHODS Salmonella cell suspension at 10^5 cfu/ml was added into either TSB or PBS containing either 9% or 24% of the extracts prepared by two different methods. Surviving Salmonella cells were enumerated after treatment for 5 h, 10 h, 24 h at 25 ℃, and reduction of Salmonella population in relation to the control was subsequently determined.

RESULTS

Results showed that the reduction of Salmonella population in TSB was significantly higher (P≤0.05) than that in PBS. Salmonella Enteritidis had a significantly lower (P≤0.05) reduction than Salmonella Tennessee in PBS and a numerically lower (P > 0.05) reduction in TSB. The extracts from the three powdered peels were significantly more effective (P≤0.05) in reducing the Salmonella population (1.67 - 2.03 Log CFU/ml) than the extract from the whole peel (1.05 Log CFU/ml) and juice powder (0.94 Log CFU/ml). Higher dose of extracts resulted in a greater reduction in Salmonella population in TSB (3.02 Log CFU/ml) or PBS (0.66 Log CFU/ml) than media with lower dose of extracts. The level of Salmonella population reduction correlated positively with the polyphenolic content (R2 0.66 - 0.99) and titratable acidity (R2 0.72 - 0.98) in the treatment systems.

CONCLUSIONS

The study suggests that the pomegranate peels have the potential to be use as preservatives in human food or animal feed to control pathogens like Salmonella.





 

Digital Health

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.





 

Medical Technology and Devices

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.





 

Molecular and Biological Research

Identification of early adopters for the early glycation products, a potential dietary supplement


Tai Guo University of Georgia BACKGROUND Early glycation products (EGPs) generated in the first two steps of Maillard reaction/glycation are proteins modified with reducing sugar moieties. This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. Our preclinical studies have found that EGPs derived from whey protein isolate (WPI) and glucose were anti-inflammatory and implied a nutraceutical application. METHODS To identify the customer segments, different hypotheses were put forward, and customer interviews were conducted either in person (wearing masks), by phone or virtually using Zoom meeting. RESULTS

Eight people were interviewed based on the initial hypothesis that individuals interested in dietary supplements would buy EGPs because this product might make them healthier. We asked questions such as: How do you feel about a new dietary supplement that makes your immune system healthier? The responses were mostly negative, including that there was no need for a dietary supplement as long as you ate healthy. Our preclinical studies showed that EGPs protected mice against hyperglycemia through altering immune balance; therefore, we put forward our second hypothesis that, unlike others, our product was to meet the objectives of diabetic patients. We have interviewed additional ten individuals who either have diabetes themselves or know someone having diabetes. We asked questions such as: Do you have any concerns or unmet needs that can't be solved by your doctors? Although most of the responses were not supportive of our hypothesis, one response was intriguing: Supplements that help with recovery from inflammation are hugely popular among serious athletes. Further research found that too many hard workouts can lead to too much inflammation (Pain) in athletes, which may result in poor athletic performances, a compromised immunity and missed practices or competitions. In addition, it was found that the global whey protein usage in the sports nutrition market was $1.6 billion in 2014 and expected to reach almost $2.7 billion in 2020. The North American market was $1.0 billion in 2014 and expected to reach $1.7 billion in 2020. The global whey protein usage in the pharmaceutical and clinical nutrition market generated $484.5 million in 2014 and was expected to reach $980.9 million in 2020, while the North American market was $309 million in 2014 and expected to reach $627 million in 2020. Our product EGPs can reduce inflammation in addition to supplementing nutritious proteins and energy, which would be an ideal and healthy dietary supplement for athletes.

CONCLUSIONS

We have identified potential early adopters for EGPs, which will be tested out using our new hypothesis that athletes buy the dietary supplement (EGPs) because it can reduce inflammation and promote post workout recovery (supported by NIH R41DK121553, and in part by NSF I-Corps funding).




1-Hexene Monooxygenase as a Biological Alternative to Delay Ripening in Climacteric Fruits


Marilyn Millett Georgia State University BACKGROUND Problems with food production and distribution are commonly discussed in the popular press and in academic journals. According to the US Department of Agriculture, 40% of all crops produced in the US are lost before reaching consumer markets. A significant contributor to this is the overripening of crops in transit to market. Commonly used methods to prevent this include refrigeration or harsh chemicals. One example of such a chemical is 1-methylcyclopropine (MCP) which breaks down ethylene and slows the Yang Cycle in climacteric fruits such as bananas, thus delaying their ripening. The Yang Cycle is a metabolic pathway where ethylene is produced via the fruit that results in the ripening of climacteric fruits. However, there are biological alternatives that break down ethylene and prevent fruit ripening without the use of harsh chemicals. The microorganism Rhodococcus rhodochrous DAP 96253 is known to possess the enzyme 1-Hexene Monooxygenase (1-HMO), commonly known as an Alkene Monooxygenase (AMO), which is believed to delay fruit ripening by converting the ethylene given off by the fruit into its epoxide form. METHODS A 1X mR3A media was used to generate induced cells of R. rhodochrous DAP 96253 in a 4L flask. After four days of growth at 30°C, the cells were then harvested via centrifugation into cell paste. In whole cell experiments, 3g paste was added to sealed plastic containers with four bananas in each container. Pictures were taken every four days for 14 days. The cells are lysed via sonication and the enzyme was purified via anion exchange chromatography (AEC). Purified product was then added to fresh bananas and observed for 14 days. RESULTS

Enzymatic activity was checked in whole cells using a color change reaction and read at 600 nm. After AEC, a BCA assay was performed to verify presence of protein.

CONCLUSIONS

This experiment will demonstrate that 1-HMO is extractable and purifiable from whole cells of R. rhodochrous while maintaining activity resulting in the delayed ripening of bananas.




Hygiene Status of Fresh Peach Packing Lines in Georgia


Peien Wang The University of Georgia BACKGROUND

Current Produce Safety Rules in the U.S. requires fresh produce packing equipment be clean and maintain sanitary conditions. This study surveyed the hygiene status of selected fresh peach packing lines in Georgia.

METHODS

Surface swabbing samples (n = 464) were collected from 14 selected sites on each of the 4 fresh peach packing lines at 3 times of a day, before packing in the morning (AM), at lunchtime (NOON), and the end of the day (PM), in 3 repeated visits per line during the harvest seasons of 2018 and 2019. Each swab sample was collected from a 100 cm2 area using a sterile sponge pre-moistened with Dey-Engley neutralizing broth. Collected samples were transported to our laboratory under refrigeration condition and analyzed within 16 - 24 hours. The levels of three hygiene indicators, total aerobes (TA), total yeasts and molds (YM), and total coliforms (TC), as well as the incidences of thermo-tolerant coliforms (TTC) and enterococci (EC) in collected samples were determined. Results were analyzed using the split-plot ANOVA tests fitted in a general linear regression model.

RESULTS

Counts of the three hygiene indicators in the AM samples were significantly lower (P<0.05) than those in the NOON and PM samples. The incidence of TTC increased from 13.50% at AM to 45.83% at PM, while that of EC from 6.75% to 15.11%. Higher levels of TA, YM, and TC counts and higher incidences of TTC and EC were observed on the brushes/rollers inside the washer/waxer and optical sizer compared to other sites sampled. In comparison, harvest bins were only high in TA and YM counts. Samples from manual sorting area had the highest TTC incidence among samples from all surveyed sites.

CONCLUSIONS

Results suggest that microbial build-up on fresh peach lines is time-dependent. The study pinpointed critical sanitation control points on fresh peach packing lines.




Inhibitory activity of aqueous extracts of pomegranate peels and juice powder on Salmonella enterica subspecies enterica serotype Tennessee and Enteritidis


Weifan Wu

The University of Georgia BACKGROUND

Pomegranate is a nutrient dense fruit rich in polyphenols including a high concentration of ellagitannins that have antimicrobial properties. Inedible pomegranate peel is reported to have even higher amounts of polyphenols than the edible arils, which makes pomegranate peel a promising source of natural preservatives.This study evaluated the inhibitory effect of aqueous extracts of different pomegranate products, including three dry peel powders, a whole peel, and a juice powder against two different strains of Salmonella enterica in tryptic soy broth (TSB) or phosphate buffered saline (PBS).

METHODS Salmonella cell suspension at 10^5 cfu/ml was added into either TSB or PBS containing either 9% or 24% of the extracts prepared by two different methods. Surviving Salmonella cells were enumerated after treatment for 5 h, 10 h, 24 h at 25 ℃, and reduction of Salmonella population in relation to the control was subsequently determined.

RESULTS

Results showed that the reduction of Salmonella population in TSB was significantly higher (P≤0.05) than that in PBS. Salmonella Enteritidis had a significantly lower (P≤0.05) reduction than Salmonella Tennessee in PBS and a numerically lower (P > 0.05) reduction in TSB. The extracts from the three powdered peels were significantly more effective (P≤0.05) in reducing the Salmonella population (1.67 - 2.03 Log CFU/ml) than the extract from the whole peel (1.05 Log CFU/ml) and juice powder (0.94 Log CFU/ml). Higher dose of extracts resulted in a greater reduction in Salmonella population in TSB (3.02 Log CFU/ml) or PBS (0.66 Log CFU/ml) than media with lower dose of extracts. The level of Salmonella population reduction correlated positively with the polyphenolic content (R2 0.66 - 0.99) and titratable acidity (R2 0.72 - 0.98) in the treatment systems.

CONCLUSIONS

The study suggests that the pomegranate peels have the potential to be use as preservatives in human food or animal feed to control pathogens like Salmonella.





 

Nanotechnology

Needle-Free Microneedle based Microparticulate Vaccine for Gonorrhea


Priyal Bagwe Mercer University, Center for Drug Delivery Research, Vaccine Nanotechnology Laboratory, College of Pharmacy BACKGROUND Neisseria gonorrhoeae is the bacteria causing gonorrhea infection and has gradually developed antimicrobial resistance. This study aims to investigate the immunogenicity of novel whole-cell inactivated gonococcal microparticulate vaccine formulation loaded in dissolving microneedles for skin delivery. METHODS N. gonorrhoeae was grown on GC agar and pileated colonies were used for bulk production in GC broth. The culture was formalin-fixed, gonococcal pellets were harvested, washed and saved as dense suspension at -80°C. The matrix for vaccine particles contained pre-crosslinked bovine serum albumin and microparticles were prepared using Buchi Mini Spray Dryer B-290. Similarly, adjuvants (Alum, MF59®) particles were prepared. The dry microparticles with 20% antigen-loading were mixed with Hyaluronic acid and Trehalose to form an aqueous solution. This solution was then loaded into the microneedle molds. PVA backing layer was later added to the partially dried microneedles in the molds. Scanning Electron Microscopy (SEM) was carried out to observe surface morphology and formation of microparticles and microneedles. The efficacy of vaccine formulation was assessed in vivo using female mice. The mice were immunized with a prime dose at week 0 followed by two boosters at weeks 2 and 4. Enzyme linked immunosorbent assay (ELISA) was used to measure immunoglobulin levels in collected mice sera. RESULTS Formalin-fixed gonococci were intact in their native form (not lysed or degraded) before processing into particulate vaccine as observed by scanning electron microscopy. This helps preserve and present all the possible antigenic epitopes in their native form to the antigen presenting cells. The average size of the particles ranged from 3.5 ± 1.2 µm. The average percent yield was found to be 85% after spray drying. The surface charge was found to be 7.1 ± 1.4 mV. The average length of microneedles as observed by scanning electron microscopy was 350 µm. The mechanical strength of the microneedles was determined by their ability to penetrate a validated skin model Parafilm M®, inserting a depth of between 300 and 400 µm. ELISA demonstrated significantly higher serum IgG, IgG1 and IgG2a titers in groups receiving adjuvanted gonorrhoea particulate vaccine (Vaccine + Adjuvants) when compared to the untreated group. CONCLUSIONS The particulate vaccine allows better uptake of antigen facilitated by the APCs causing improved antigen presentation and subsequent immune response by activation of T cells. Skin delivery of the inactivated whole-cell gonococcal microparticulate vaccine formulation loaded in hyaluronic-acid based dissolving microneedles is therefore an effective vaccination strategy.




Moving Towards a Universal Flu Vaccine: Assessment of In Vitro Immunogenicity and Antibody Levels of a Subunit Microparticle Influenza Vaccine


Keegan Braz Gomes Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy BACKGROUND The 2019-2020 influenza season was amongst the most deadly flu seasons within the last decade, resulting in tens of thousands of deaths in the United States. The aim of the proposed research was to investigate a universal vaccine using a highly conserved influenza ectodomain matrix-2 protein (M2e) virus-like particles (VLPs). The M2e VLP and two adjuvants were incorporated into a microparticulate matrix. The vaccine (M2e + adjuvants microparticles) was administered via the transdermal route of administration using ablative laser to explore a minimally invasive method of administration. METHODS The M2e VLP and adjuvants (Alhydrogel® and MPL-A®) were encapsulated into a biodegradable polymer matrix and spray dried to produce antigen and adjuvant-loaded microparticles. The particles were assessed for their in vitro immunogenicity (ability to stimulate immune cells) and cytotoxicity (toxicity to mammalian cells). Innate immunogenicity was determined by assessing nitric oxide production. The expression of antigen-presenting molecules was evaluated in antigen-presenting cells (APCs) pulsed with antigen and adjuvant microparticles. The vaccine (M2e + adjuvants microparticles) was administered via the transdermal route of administration in a preclinical murine model using ablative laser to form micropores in the skin. Throughout the study, M2e-specific immunoglobulin G (IgG) in the serum samples collected from the mice was assessed using Enzyme-Linked Immunosorbent Assay (ELISA). At Week 12, the mice were challenged with live influenza (A/Philippines/2/82 virus strain). RESULTS The vaccine microparticles produced high levels of nitric oxide in vitro indicating a strong innate immune response potential. The M2e microparticles were also shown to be non-cytotoxic in a range of concentrations for up to 4 days in vitro. APCs pulsed with the vaccine showed significantly higher levels of antigen-presenting molecules: major histocompatibility complex I (MHC I), MHC II, CD40, and CD80. The mice vaccinated with M2e VLP + adjuvants microparticles demonstrated elevated levels of IgG antibodies, beginning at week 7. The IgG levels in vaccinated groups were maintained through week 10 and were significantly higher compared to the unvaccinated groups. CONCLUSIONS

We developed an efficacious flu vaccine comprising of M2e VLP and adjuvants in a microparticle matrix. The formulated microparticulate vaccine was found to be immunogenic and easy to administer via the transdermal route of administration. In the future, alternate routes for vaccine delivery, such as fast-dissolving microneedles, will be explored.




A Prophylactic Microparticulate Vaccine for Needle-free Immunization against Zika


Akanksha Kale Mercer University College of Pharmacy BACKGROUND The world witnessed a zika outbreak in the year 2015-2016, with the WHO declaring it as a public health emergency. Zika fever is a zoonotic disease transmitted to humans through bites of mosquitoes of the Aedes aegyptii species. Apart from mosquito bites, the virus can also be transmitted through sex, blood transfusions, and from mother to fetus during pregnancy. Epidemiologically, zika has been associated with microcephaly and Guillain-Barré Syndrome. However, no approved treatment or vaccine is available for zika thus far. Our proposed approach focuses on formulating a microparticulate vaccine using inactivated zika virus (strain PRVABC59) as the antigen. Subsequently, the microparticulate vaccine is loaded into dissolving microneedle patches and administered via the transdermal route in a pre-clinical murine model. METHODS

The inactivated zika virus-loaded microparticles were formulated by a double emulsion solvent evaporation method using poly(lactic-co-glycolic) acid as the polymer. The microemulsion was lyophilized to obtain the dried vaccine microparticles. Adjuvanted microparticles containing a combination of Alhydrogel® and MPL-A® were formulated following a similar method. The microparticles were characterized for size, morphology, surface charge and encapsulation efficiency. Vaccine microparticles were loaded in the dissolving microneedle patches. Antigen integrity was determined using SDS-PAGE. Griess' assay was performed in murine dendritic cells to determine the ability of a microparticulate vaccine to induce the production of nitric oxide. The cytotoxicity of microparticles was determined using MTT assay.
Microparticulate vaccine with and without adjuvants were administered via the intramuscular (i.m.) as well as the transdermal routes of administration to Swiss Webster Mice. Serum samples were collected every two weeks and assessed for IgM and IgG antibody levels.

RESULTS The particle size of microparticles was 573.4 ± 10.18 nm with a polydispersity index of 0.294 ± 0.133. The zeta potential of microparticles was found to be -22.6 ± 0.503 mV. The encapsulation efficiency was in the range of 55-70%. Scanning electron microscopy images showed that the microparticles are spherical. Dendritic cells pulsed with the microparticulate zika vaccine produced significantly higher amount of nitrite which is a marker of innate immunity as compared to the commercial measles vaccine. The microparticulate vaccine was non-cytotoxic as compared to dimethyl sulfoxide, a known cytotoxic agent, as a control. SDS-PAGE showed that formulation process does not affect the antigen. A robust humoral immune response is essential to neutralize the infectious pathogen. The mice immunized with adjuvanted microparticulate vaccine via both intramuscular and transdermal routes produced significant antibody titers when compared to unvaccinated mice. CONCLUSIONS

The microparticulate zika vaccine was found to stimulate innate immune response in vitro. The in vivo immunization via both i.m. as well as transdermal route revealed that the microparticulate vaccine was able to generate a humoral immune response in a pre-clinical murine model.




Deploying a Th1 Polarized Immune Response Against RSV Using Laser Assisted Transdermal Delivery of a Microparticulate Vaccine


Ipshita Menon Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy BACKGROUND Prior attempts to use the formalin-inactivated form of the virus resulted in the tragic failure of the vaccine. As a result, there is no licensed vaccine for RSV thus far. Virus-like particles formed using the F protein (F-VLP) is a highly immunogenic albeit non- virulent antigen. The presentation of exogenous antigens by antigen-presenting cells (APCs) is enhanced by encapsulating them in a biodegradable polymer matrix. RSV is an infectious disease that majorly affects infants and children, hence, a "needle-free" approach for immunization will be favorable. The objective of this study was to evaluate the immunogenicity of F-VLP microparticle (MP) along with monophosphoryl lipid A (MPL) MP administered via the transdermal route using Precision Laser Epidermal System (P.L.E.A.S. E®). METHODS

Vaccine-adjuvant combination was administered to Swiss Webster mice via the transdermal route. The mice sera were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine immunoglobulin G (IgG) and the subtypes. The mice were then challenged with RSV A2 virus at week 13. Post challenge, the immune-organs and lungs was assessed for the expression of T and B cell subtypes by flowcytometry. In addition, the lung homogenates were analyzed for levels of IgG and subtypes as well as IgA by ELISA. Furthermore, the viral load in the lungs was analyzed using an immune plaque assay.

RESULTS The immune sera as well as lung homogenates of mice immunized with F-VLP MP + MPL MP had significantly high levels of IgG and IgG2a. The induced immune response seemed to be skewed towards the (Th)1 arm which is essential in fighting an infectious disease. The mice immunized with F-VLP MP + MPL MP elicited a higher CD8+ and CD4+cell count in lymph node and spleen cell populations when compared with the mice immunized with FI RSV as well as naïve mice. They also had significantly higher expression of IFN-γ in the spleen cells. The ELISA analysis of the lung homogenates results revealed significantly higher levels IgA in the mice immunized with F-VLP MP + MPL MP. IgA is a marker of mucosal immunity and confers protection against RSV. The immune plaque assay proved that the mice immunized with F-VLP MP + MPL MP had negligible viral plaques as compared to mice immunized with formalin inactivated RSV. CONCLUSIONS

A balanced induction of cellular as well as humoral immune response was observed. (Th)1 polarized immune response is vital to combat a viral infection like RSV and avoiding ERD. The transdermal vaccine was able to confer protection against RSV as represented by high levels of IgA in the lungs and negligible lung viral loads as well. Thus, the novel F-VLP MP + MPL MP vaccine, administered via the transdermal route using ablative laser shows promise as a vaccine for RSV.




Combat-ready for COVID-19: Pain-free, SARS Spike S1 Recombinant Protein Microparticulate Vaccine Administered using Microneedles


Smital Rajan Patil Mercer University College of Pharmacy BACKGROUND The rampancy of COVID-19 has affected around 32.3 million people and caused more than 984,000 deaths worldwide. Considering the highly contagious nature of the virus, the need for a safe and efficacious vaccine for COVID-19 is highly critical. The SARS-CoV spike S1 receptor binding domain (RBD) protein is a viral epitope that is capable of inducing an immune response in the body and thus a suitable antigen for formulating a coronavirus vaccine. Additionally, microparticles (MPs) are suitable delivery vehicles for vaccine antigens as they are better taken up by antigen-presenting cells, inducing a more robust immune response against the antigen. For this study, the spike RBD protein was loaded into poly (lactic-co-glycolic acid) (PLGA) MPs, which were then incorporated into dissolving microneedles, which have shown to be a promising delivery system for large molecules such as proteins. METHODS

Spike RBD protein-loaded MPs (SPMPs) were formulated via a double emulsion method. The microparticles were subsequently lyophilized, characterized, and assessed for innate and adaptive immune response in vitro. Vaccine microparticles were loaded into a hyaluronic acid gel and centrifuged to produce vaccine-loaded fast-dissolving microneedles arrays. The efficacy of the vaccine microneedle patches will be assessed in vivo in a preclinical murine model.

RESULTS The SPMPs were successfully formulated and characterized for their size, surface morphology, charge, and antigen encapsulation efficiency. The vaccine particles induced a significantly higher nitrite production in mammalian cells compared to non-treated cells and cells treated with antigen unloaded microparticles. Dendritic cells pulsed with the vaccine microparticles also produced significantly higher expression of antigen presenting molecules: major histocompatibility complex I (MHC I), CD80, MHC II, and CD40 on the surface of the dendritic cells. CONCLUSIONS

This formulated vaccine thus shows high immunogenicity in vitro and has the potential to produce a robust immune response in a murine model, conferring long-lasting protection against coronavirus. This has a potential to be promising vaccine in the ongoing COVID-19 pandemic the world is facing currently.




Fighting Coronaviruses - A microparticulate vaccine microneedle patch against coronaviruses


Sharon Vijayanand Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy BACKGROUND

Coronaviruses are zoonotic viruses known to cause mild to serious upper respiratory tract illnesses in both animals as well as humans. The causal agent of COVID-19, the SARS CoV-2 is a novel pandemic strain, which has caused global chaos. Consequently, there exists an immediate need for an effective vaccine that is reproducible, safe, and scalable. This study aims to test the immunogenicity of a novel heat-inactivated coronavirus microparticulate (MP) vaccine administered via dissolving microneedles (MN). The vaccine formulation utilizes a microparticulate matrix that provides protection of the antigen and improved antigen uptake in antigen-presenting cells. Furthermore, the addition of adjuvants - alum and MF59, can enhance the overall immune response to the antigen. To enhance patient compliance and to eliminate painful needle administration, a novel transdermal route of administration via microneedles was chosen for delivering coronavirus antigen in a microparticulate matrix.

METHODS

Formulating the microparticulate vaccine involved the use of a double emulsion method and lyophilization to produce antigen and adjuvant poly (lactic-co-glycolic acid) (PLGA) MPs. The ability of the vaccine MPs to elicit an in vitro innate immune response was assessed using a nitric oxide assay. The in vitro antigen presentation of the microparticulate vaccine by the antigen-presenting cells (APCs) was assessed by measuring the expression of major histocompatibility complexes I/II (MHC I/II) and their co-stimulatory molecules CD 80/40 on the surface of dendritic cells using flow cytometry. For in vivo assessment, the mice were immunized with the MP vaccine loaded dissolving MN patches, formulated using Hyaluronic acid (HA). A prime, followed by two booster doses of vaccine were administered to the mice at weeks 0, 2, and 4 respectively. Sera of mice were collected at various points to determine the antibody titers following dosing. Following sacrifice, immune organs will be isolated and analyzed for the expression of immune markers.

RESULTS The MPs were less than 1 microns in size. The Griess's nitrite assay showed a much higher release of nitric oxide (NO) by dendritic cells pulsed with the microparticulate vaccine as compared to untreated cells. Flow cytometry analysis confirmed much higher expression of antigen presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 respectively on the surface of APCs. CONCLUSIONS

The microparticulate vaccine produces an effective innate and adaptive immune response as indicated by the significant release of nitric oxide and expression of antigen-presenting molecules by the APCs. Additionally, the microparticulate vaccine enhances the immunogenicity of the heat-inactivated coronavirus, by conferring cross-presentation of the antigen on the surface of the APCs. Further analysis of serum and T-cell phenotypes from immune organs in mice will confirm if the vaccine can produce a robust immune response.





 

Regulatory Affairs

Identification of early adopters for the early glycation products, a potential dietary supplement


Tai Guo University of Georgia BACKGROUND Early glycation products (EGPs) generated in the first two steps of Maillard reaction/glycation are proteins modified with reducing sugar moieties. This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. Our preclinical studies have found that EGPs derived from whey protein isolate (WPI) and glucose were anti-inflammatory and implied a nutraceutical application. METHODS To identify the customer segments, different hypotheses were put forward, and customer interviews were conducted either in person (wearing masks), by phone or virtually using Zoom meeting. RESULTS

Eight people were interviewed based on the initial hypothesis that individuals interested in dietary supplements would buy EGPs because this product might make them healthier. We asked questions such as: How do you feel about a new dietary supplement that makes your immune system healthier? The responses were mostly negative, including that there was no need for a dietary supplement as long as you ate healthy. Our preclinical studies showed that EGPs protected mice against hyperglycemia through altering immune balance; therefore, we put forward our second hypothesis that, unlike others, our product was to meet the objectives of diabetic patients. We have interviewed additional ten individuals who either have diabetes themselves or know someone having diabetes. We asked questions such as: Do you have any concerns or unmet needs that can't be solved by your doctors? Although most of the responses were not supportive of our hypothesis, one response was intriguing: Supplements that help with recovery from inflammation are hugely popular among serious athletes. Further research found that too many hard workouts can lead to too much inflammation (Pain) in athletes, which may result in poor athletic performances, a compromised immunity and missed practices or competitions. In addition, it was found that the global whey protein usage in the sports nutrition market was $1.6 billion in 2014 and expected to reach almost $2.7 billion in 2020. The North American market was $1.0 billion in 2014 and expected to reach $1.7 billion in 2020. The global whey protein usage in the pharmaceutical and clinical nutrition market generated $484.5 million in 2014 and was expected to reach $980.9 million in 2020, while the North American market was $309 million in 2014 and expected to reach $627 million in 2020. Our product EGPs can reduce inflammation in addition to supplementing nutritious proteins and energy, which would be an ideal and healthy dietary supplement for athletes.

CONCLUSIONS

We have identified potential early adopters for EGPs, which will be tested out using our new hypothesis that athletes buy the dietary supplement (EGPs) because it can reduce inflammation and promote post workout recovery (supported by NIH R41DK121553, and in part by NSF I-Corps funding).




1-Hexene Monooxygenase as a Biological Alternative to Delay Ripening in Climacteric Fruits


Marilyn Millett Georgia State University BACKGROUND Problems with food production and distribution are commonly discussed in the popular press and in academic journals. According to the US Department of Agriculture, 40% of all crops produced in the US are lost before reaching consumer markets. A significant contributor to this is the overripening of crops in transit to market. Commonly used methods to prevent this include refrigeration or harsh chemicals. One example of such a chemical is 1-methylcyclopropine (MCP) which breaks down ethylene and slows the Yang Cycle in climacteric fruits such as bananas, thus delaying their ripening. The Yang Cycle is a metabolic pathway where ethylene is produced via the fruit that results in the ripening of climacteric fruits. However, there are biological alternatives that break down ethylene and prevent fruit ripening without the use of harsh chemicals. The microorganism Rhodococcus rhodochrous DAP 96253 is known to possess the enzyme 1-Hexene Monooxygenase (1-HMO), commonly known as an Alkene Monooxygenase (AMO), which is believed to delay fruit ripening by converting the ethylene given off by the fruit into its epoxide form. METHODS A 1X mR3A media was used to generate induced cells of R. rhodochrous DAP 96253 in a 4L flask. After four days of growth at 30°C, the cells were then harvested via centrifugation into cell paste. In whole cell experiments, 3g paste was added to sealed plastic containers with four bananas in each container. Pictures were taken every four days for 14 days. The cells are lysed via sonication and the enzyme was purified via anion exchange chromatography (AEC). Purified product was then added to fresh bananas and observed for 14 days. RESULTS

Enzymatic activity was checked in whole cells using a color change reaction and read at 600 nm. After AEC, a BCA assay was performed to verify presence of protein.

CONCLUSIONS

This experiment will demonstrate that 1-HMO is extractable and purifiable from whole cells of R. rhodochrous while maintaining activity resulting in the delayed ripening of bananas.




Hygiene Status of Fresh Peach Packing Lines in Georgia


Peien Wang The University of Georgia BACKGROUND

Current Produce Safety Rules in the U.S. requires fresh produce packing equipment be clean and maintain sanitary conditions. This study surveyed the hygiene status of selected fresh peach packing lines in Georgia.

METHODS

Surface swabbing samples (n = 464) were collected from 14 selected sites on each of the 4 fresh peach packing lines at 3 times of a day, before packing in the morning (AM), at lunchtime (NOON), and the end of the day (PM), in 3 repeated visits per line during the harvest seasons of 2018 and 2019. Each swab sample was collected from a 100 cm2 area using a sterile sponge pre-moistened with Dey-Engley neutralizing broth. Collected samples were transported to our laboratory under refrigeration condition and analyzed within 16 - 24 hours. The levels of three hygiene indicators, total aerobes (TA), total yeasts and molds (YM), and total coliforms (TC), as well as the incidences of thermo-tolerant coliforms (TTC) and enterococci (EC) in collected samples were determined. Results were analyzed using the split-plot ANOVA tests fitted in a general linear regression model.

RESULTS

Counts of the three hygiene indicators in the AM samples were significantly lower (P<0.05) than those in the NOON and PM samples. The incidence of TTC increased from 13.50% at AM to 45.83% at PM, while that of EC from 6.75% to 15.11%. Higher levels of TA, YM, and TC counts and higher incidences of TTC and EC were observed on the brushes/rollers inside the washer/waxer and optical sizer compared to other sites sampled. In comparison, harvest bins were only high in TA and YM counts. Samples from manual sorting area had the highest TTC incidence among samples from all surveyed sites.

CONCLUSIONS

Results suggest that microbial build-up on fresh peach lines is time-dependent. The study pinpointed critical sanitation control points on fresh peach packing lines.




Inhibitory activity of aqueous extracts of pomegranate peels and juice powder on Salmonella enterica subspecies enterica serotype Tennessee and Enteritidis


Weifan Wu

The University of Georgia BACKGROUND

Pomegranate is a nutrient dense fruit rich in polyphenols including a high concentration of ellagitannins that have antimicrobial properties. Inedible pomegranate peel is reported to have even higher amounts of polyphenols than the edible arils, which makes pomegranate peel a promising source of natural preservatives.This study evaluated the inhibitory effect of aqueous extracts of different pomegranate products, including three dry peel powders, a whole peel, and a juice powder against two different strains of Salmonella enterica in tryptic soy broth (TSB) or phosphate buffered saline (PBS).

METHODS Salmonella cell suspension at 10^5 cfu/ml was added into either TSB or PBS containing either 9% or 24% of the extracts prepared by two different methods. Surviving Salmonella cells were enumerated after treatment for 5 h, 10 h, 24 h at 25 ℃, and reduction of Salmonella population in relation to the control was subsequently determined.

RESULTS

Results showed that the reduction of Salmonella population in TSB was significantly higher (P≤0.05) than that in PBS. Salmonella Enteritidis had a significantly lower (P≤0.05) reduction than Salmonella Tennessee in PBS and a numerically lower (P > 0.05) reduction in TSB. The extracts from the three powdered peels were significantly more effective (P≤0.05) in reducing the Salmonella population (1.67 - 2.03 Log CFU/ml) than the extract from the whole peel (1.05 Log CFU/ml) and juice powder (0.94 Log CFU/ml). Higher dose of extracts resulted in a greater reduction in Salmonella population in TSB (3.02 Log CFU/ml) or PBS (0.66 Log CFU/ml) than media with lower dose of extracts. The level of Salmonella population reduction correlated positively with the polyphenolic content (R2 0.66 - 0.99) and titratable acidity (R2 0.72 - 0.98) in the treatment systems.

CONCLUSIONS

The study suggests that the pomegranate peels have the potential to be use as preservatives in human food or animal feed to control pathogens like Salmonella.





 
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