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Oral Presentations

Ph.D. Student/Postdoc Oral Presentations

POP01

Exosome Biogenesis: Implications in Cardiovascular Disease Progression and Time of Death

Courtnee’ R. Bell1, Sparkle D. Williams2, Brian Sims3, Glenn C. Rowe4, and Qiana L. Matthews1,*

1Microbiology Program, Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104; 2Departments of Pediatrics, Neurobiology and Cell, Developmental and Integrative Biology, Division of Neonatology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3Department of Medicine, 4Cardiovascular Diseases, University of Alabama at Birmingham, Birmingham, Alabama 35294, *Email: qmatthews@alasu.edu

Cardiovascular disease (CVD) refers to conditions that involve the narrowing or blockage of blood vessels that can potentially lead to the onset of a heart attack, and may be the cause of sudden, suspicious, or unnatural death in criminal cases. CVDs are frequently concealed and discovered only at postmortem autopsy through the use of molecular investigations. Determining cause of death, and time since death, using conventional autopsy techniques relies on interpretation of physical changes that occur after death. Although common in their use, these physical changes cannot be documented in cases of advanced or prolonged decomposition.  The need for more accurate alternative methods of postmortem investigation has sparked interest in the gene expression shifts that take place after death. The goal of this project is to identify potential exosomal markers for the identification of sudden cardiac death and determination of time of death.

Currently, we are quantifying the effect that alcohol administration has on exosomes biogenesis of human cardiomyocyte cell line, AC16.  The AC16 cell line was cultured in exosome-free medium and was either not treated (control) or treated with 50 mM or 100mM of alcohol for 24, 48, or 72 h.  AC16 cell viability was significantly decreased with treatments of 50 mM or 100 mM of alcohol. The presence of exosomes was confirmed using NanoSight analysis, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). The groups of exosomes were isolated using a series of high-speed ultracentrifugation and quantitated using the Lowry dilution method.   By means of ELISA, various tetraspanins (CD9, CD63, CD81) were confirmed to be present on AC16-derived exosomes.  Our findings show that alcohol administration on AC16 cell line significantly impacts actin and heat shock protein 70 within AC16-derived exosomes. In this study, we are also evaluating postmortem exosomes from mice and swine samples, at 0hr time of death.  Blood was isolated and exosomes were extracted and confirmed by means of NanoSight, SDS-PAGE and ELISA. Our findings, identified a baseline of serum-derived exosome associated proteins (clathrin, actin, tubulin) that can be used in postmortem analysis in mouse and swine and animal model systems at a 0hr time of death. The evaluation of the impact of alcohol on cardiac cell death and postmortem exosome biogenesis will yield new insight into the importance of exosomes in a variety of physiological and pathological settings including cardiovascular disease. Our future studies will include evaluating exosomes at other times of death as well as evaluating postmortem gene expression.

POP2

The Postmortem Clostridium Effect in Human Decomposition

Sheree J. Finley1 and Gulnaz T. Javan2,*

1Forensic Science Program, Department of Physical/Forensic Sciences, Alabama State University, Montgomery, Alabama 36104; 2Department o Physical/Forensic Sciences, Alabama State University, Montgomery, Alabama 36104. *Email:gjavan@alasu.edu

Human thanatomicrobiome studies have demonstrated that the predominant putrefactive bacteria within internal organs of decaying bodies at short times of death are obligate anaerobes, Clostridium spp. (Javan et al., 2016a; Javan et al., 2016b). Although Clostridium is ubiquitous in internal organs of decaying corpses, there are large gaps in our knowledge of the role that this bacteria contributes at long times since death. We performed phylogenetic surveys of thanatomicrobiome communities in livers and spleens of 45 cadavers from criminal casework. Amplicons of the 16S rRNA gene V4 hypervariable region and V3-4 conjoined regions were sequenced on the Illumina MiSeq platform. The results of this first-of-its-kind study implicated Clostridium spp. as the primary mediators of putrefaction in internal organs at long postmortem intervals (up to 10 days). The largest order of bacteria was Clostridiales and seven of the most abundant species were Clostridium spp. This groundbreaking research also introduced and described a novel scientific concept, the Postmortem Clostridium Effect (PCE), which pinpoints some of the roles that Clostridium spp. play during human putrefaction (Javan et al. 2017). The study encompassed one of the largest cohort of cadavers to date and added empirical data to the Human Postmortem Microbiome Project. Findings of our previous thanatomicrobiome studies discovered that a majority of microorganisms in the human body at short times after death were Clostridia. However, we have not now demonstrated that Clostridium spp. also predominate at long times since death. These finding provide insightful information concerning the omnipresence and persistence of Clostridia during human putrefaction.

Javan GT, Finley SJ, Abidin Z, and Mulle JG (2016a) The thanatomicrobiome: A missing piece of the microbial puzzle of death. Frontiers in Microbiology, 7.

Javan GT, Finley SJ, Can I, Wilkinson JE, Hanson JD, and Tarone AM (2016b) Human thanatomicrobiome succession and time since death. Scientific reports, 6, 29598.

Javan GT, Finley SJ, Smith T, Miller J and Wilkinson JE (2017) Cadaver thanatomicrobiome signatures: the ubiquitous nature of Clostridium species in human decomposition. Frontiers in Microbiology, 8, 2096.

POP03

Ethanol Exposure Impacts Exosome Biogenesis of HeLa Cell-derived Exosomes

Leandra B. Jones1, Jayde S. Price2, Sparkle D. Williams3, Alexandre Krendelchtchikov3,

Brian Sims3, and Qiana L. Matthews1,*

1Microbiology Program, Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104; 2Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104; 3Departments of Pediatrics, Neurobiology and Cell, Developmental and Integrative Biology, Division of Neonatology, University of Alabama at Birmingham, Birmingham, Alabama 35294, *Email: qmatthews@alasu.edu

Exosomes are microvesicles that range from 30 – 100 nm in size. They can be found in various bodily fluids (breast milk, blood, semen, urine) and function mainly in cell-to-cell communication. Exosomes allow intracellular communication via DNA, RNA, and protein trafficking. External stressors such as alcohol have been known to alter the biogenesis and release of these vesicles. Currently, we are quantifying alcohol’s effects on exosome biogenesis at varying time points and concentrations in HeLa (cervical cancer) cells. HeLa cell were treated with 50 mM and 100 mM of Ethanol, respectively, for 24, 48 and 72 h.  Cell viability was observed at 72 h post Ethanol treatment. HeLa cell viability was significantly decreased with treatments of 50 mM and 100 mM of Ethanol.  After treatment, exosomes were purified from media via high-speed centrifugation. Exosome quantity was determined by the Lowry protein quantitation method. NanoSight technology was used to measure exosome diameter and exosome count after alcohol exposure. Enzyme-Linked Immunosorbent Assay was performed on exosomes to detect various exosome-specific proteins.  The presence of exosomes was confirmed by the detection of various tetraspanins (CD9, CD63, CD81), as well as chaperone proteins (HSP90_, HSP70, HSP60). Our findings show that alcohol-derived exosomes contain significant quantities of HSP60, HSP70, Fas, and Caspase-9 when compared to control- derived exosomes.

POP04

The Effect of Alcohol on the Biogenesis and Composition of Microglia-Derived BV-2 Exosomes

Brennetta J. Crenshaw1, Brian Sims2, Sparkle D. Williams2, Qiana L. Matthews1,*

1Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering, and Mathematics, Alabama State University, Montgomery, Alabama 36104; 2Department of Pediatrics, Neurobiology and Cell, Developmental and Integrative Biology, Division of Neonatology, University of Alabama at Birmingham, Birmingham, Alabama 35294. *Email: qmatthews@alasu.edu

Exosomes are small extracellular vesicles that are formed during the maturation of endosomes. In recent years, exosomes have emerged as an important tool for intercellular communication through the transfer of biologically active proteins, lipids, and RNAs. They are secreted by most cell types (antigen-presenting cells, epithelial cells, glial and tumor cells) in the body and are found in biological fluids such as blood, urine, semen, saliva, cerebrospinal fluid, and breast milk. The biogenesis of exosomes changes based on external factors/stimuli.  Some factors have been shown to regulate exosome biogenesis. One substance that may be relevant is alcohol. As a small molecule, alcohol can easily cross membrane barriers and quickly be distributed to all major organs.  One notable organ that interacts with alcohol is the brain.  Alcohol interacts with brain receptors, interfering with communication between nerve cells, and suppressing the excitatory nerve pathway. The purpose of this project was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from brain microglia cells, BV2. The BV2 cell line was cultured in exosome-free medium and was either not treated (control) or treated with 50mM or 100mM of alcohol for 24, 48, and/or 72 hours. The cell morphology was examined through light microscopy. The groups of exosomes were isolated using a series of high-speed ultracentrifugation and protein quantitated using the Lowry dilution method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the exosomal proteins based on size and Coomassie blue staining was performed to identify the presence of exosomal proteins in each treatment group. Enzyme-linked immunosorbent assay (ELISA) was performed on the exosomes to confirm the presence of exosomal proteins on BV2 cells. Our studies revealed that exosome biogenesis and composition was affected by alcohol treatment. At 50mM and 100mM alcohol exposure, cells appeared to show apoptotic behavior after 48-hour and 72-hour treatments. Cells appeared to be shrunken compared to the control untreated cells and viability was decreased significantly. The average of three experiments demonstrated through fold change that alcohol treatment at 48 hours at the high dose caused a significant increase of tetraspanin CD63 expression in exosomes. In addition, alcohol treatment after 48 hours caused significant increases of heat shock protein 90 beta (HSP90_) compared to the control. At 72 hour treatment with alcohol, we observed a significant increase of cytoskeletal protein actin expression in exosomes when compared to control. More experiments will be conducted to gain additional knowledge on the impact of alcohol on exosome biogenesis in BV2 cells.

POP05

Nanoparticles Functionalized with PenicillinEffect the Viability of Multi- Antibiotic-Resistant Isolates of Streptococcus pneumonia

Veolanda A. Peoples, Shannon Williams, Ronda Bibbs, and Mamie T. Coats*

Center for NanoBiotechnology Research, Alabama State University, Montgomery, Alabama 36104. *Email: mcoats@alasu.edu

Streptococcus pneumoniae is a respiratorypathogen which remains a major cause of morbidity and mortality worldwide in spite of available vaccines and antibiotics.The development of a novel antimicrobial drug is needed in order to conquer existing mutating S.pneumoniae isolates. Our goal was to investigate the usefulness of penicillin conjugated gold nanoparticles (AuNP-Pen) and encapsulated poly lactic-co-glycolic (PLGA-Pen) nanoparticles as potential multivalent antimicrobials inhibiting the growth of clinically relevant planktonic S. pneumoniae. Gold nanoparticles (AuNP) were synthesized throughthe reductionof gold (III) chloride trihydrate (HAuCl4)withsodium borohydride (NaBH4) using the citrate reduction method.The final nanoparticle suspension yields a concentration of 2.5 x 10-4M. Penicillin was conjugated to the surface of the particles via formation of a thioether bond.  PLGA-Pen nanoparticles were prepared by emulsification-diffusion method.  All susceptibility studies were done over five hours of incubation in enriched media at 37ºC. The S. pneumoniae isolates used were isolated from pediatric patients and had varying susceptibility to penicillin. The AuNP-Pen produced were 5-10nm spherical particles with 1.03 x 10-6 molof penicillin/ng particle. While the encapsulated PLGA –Pen particles were considerable larger at 150-200nm.  Following exposure to the nanoparticles, planktonic pneumococci showed reduced viability in the presence of the 0.125ng/µl AuNP-Pen and 0.25ng/µl PLGA-Pen when compared to AuNP and PLGA.  However, independent of the level of penicillin susceptibility, there was not a significant difference in the inhibitory ability of free penicillin compared to that of AuNP-Pen and PLGA-Pen.  Furthermore, while there was a trend for the isolates to survive less well in AuNP-Pen and PLGA-Pen than AuNP and PLGA, respectively; the difference was not statistically significant.  While the tested nanoparticles were not effective against the antibiotic resistant S. pneumoniae, they will be examined against other pathogens.  Also, because the use of these nanomaterials as therapeutics (against other bacteria) is possible, the genetic response of S. pneumoniaeto the conjugate and encapsulated nanoparticles will be examined in the future. [This work was supported by NSF-CREST (HRD-1241701); NSF-PIRE-1545884; and NSF-TPAC HRD-1432991].

POP06

Microbes May Play a Significant Role in Hg Contaminant Reduction in Soils Compared to Organic Sorbents

Timothy Egbo1, Carrie Sanders1, Carrie Miller2, Alexander Josh3, Yazeed Abdelmageed1, and Boakai K. Robertson1,*

1Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104; 2Department of Biological & Environmental Sciences, Troy University, Troy, Alabama 36082; and 3Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37871. *Email: brobertson@alasu.edu

The US Environmental Protection Agency (EPA) has identified the Y-12 National Security Complex around the Oak Ridge Reservation as being heavily contaminated with mercury (Hg) specifically around the upper and lower East Fork Poplar Creek (EFPC) ecosystem. Hg contamination within the Oak Ridge Reservation has been an ongoing research. The use of sorbents has been proposed as a cleanup strategy; understanding the biological factors that might influence the efficiency of the sorbents or clean up strategy is very essential. In recent studies, we have identified Serratia marcescens to have anti-biofilm properties. The goal of this study was to use microbe (S. marcescens) isolated from site where Hg is not more than 54.5 ug/g of total Hg (HgT) dry weight to evaluate microbial influence on sorbent Organoclay™. We collected bank soils contaminated with Hg from the EFPC. The samples were collected from two distinct soil layers, the top lighted-colored soil labeled SB 14-8 downstream of the creek, the bottom dark colored soil upstream of the creek labeled SB 5-8 and uncontaminated soil from the Hinds Creek area. Pure cultures were obtained from soil sample SB 14-8 using broad-spectrum nutrient broth, and natural medium formulated using extracts from the different soil samples. S. marcescens was allowed to grow to O.D600 0.5 and then the soil was added to the culture. Culture of S. marcescens in the presence of sorbent Organoclay™ and soil was allowed to mix on a shaker for 48 h at 25oC after allowing the soil to stand in diH2O for 14 days. Organoclay™ was added to determine the amount of Hg sorbed in the presence or absence of microorganism. HgT concentrations were determined using cold vapor atomic fluorescence in accordance with EPA method 1631. Our results showed that S. marcescens significantly reduced Hg in solution compared to the sorbent; nonetheless, this increase in reduction was found to be more significant within the SB 14-8. We did not see any significant influence of S. marcescens on the sorbents compared to S. marcescens alone. Future study will examine further possible interaction of S. marcescens with biofilms formation on a sorbent. This work is supported by National Science Foundation's Alliances for Graduate Education and the Professoriate (AGEP) Program, Grant No. 1432991, and Department of Energy-Savannah River Nuclear Solution, Grant No. 0000217390.

POP07

Combination Therapy of Resveratrol and Microbial Metabolites Short-chain Fatty Acids Utilizes Cell Cycle Arrest to Induce Apoptosis

James Stokes III1, Sanjay Kumar1, Sabita Saldanha1, Karyn Scissum Gunn1, Udai P. Singh2, and Manoj Mishra1,*

1Cancer Biology Research and Training, Department of Biological Sciences, Alabama State University, Alabama 36101; 2Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29208. *Email: mmishra@alasu.edu

Short-chain fatty acids (SCFAs) are the chemical by-products of microbial fermentation. Resistant carbohydrates, some starches, and foods generally rich in fiber are used as substrates for this process. SCFAs are known to activate key cellular receptors such as the G-protein coupled receptor 43 also known as the free fatty acid receptor 2 GPR43/FFA2, which mediates programmed cell death in various cancer cells and modulate the expression of well-known tumor suppressor genes such as PTEN. Additionally, numerous studies have successfully demonstrated that SCFAs have the capacity to initiate cell cycle arrest as a mechanism to mediate said programmed cell death (apoptosis). Increased mitochondrial activity has also been linked to the SCFA-initiated apoptotic pathway; thereby, confirming the often beneficial relationship enjoyed between SCFAs and mammalian mitochondria. Resveratrol (RES) is a polyphenolic compound found in select vegetables and fruits and has been known for its versatility in preventing and reversing a number of diseases including some cancers. Much like SCFAs, RES has the ability to induce programmed cell death in many clinical cancer models via a variety of intracellular mechanisms. Briefly, in an effort to determine if SCFAs and RES combination therapy would a) induce growth arrest as a means to facilitate apoptosis in vitro and b) significantly impact mitochondrial function, transgenic adnenocarcinoma of mouse prostate (TRAMP) TRAMP C1 and C3 cell lines were used as an experimental model for testing. Cells were incubated with varying concentrations of SCFAs and RES both separately and synergistically. Post-treatment, techniques such as cell viability, cell cycle arrest, and mitochondria membrane potential (__m) assays were conducted to complete experimentation. Our result demonstrated that SCFAs and RES utilize different pathways to deliver their anti-cancer potency.

POP08

Cloning and Expression of Epsilon 34 Tailspike Gene Using the pET30a Ligation Independent Clone Vector

Joseph Ayariga, Robert Ward, Karthik Venkatesan, and Robert Villafane*

Microbiology Program, Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104. *Email: rvillafane@alasu.edu

Renewed interest within the scientific community for phage therapy has also stimulated phage research investigations which include not only the conventional identification, characterization and genomic sequencing of these unknown phages for their medical utilities but the move to engineer phage particles, or phage parts from known and previously uncharacterized phages for their therapeutic benefits. Published findings are indicative of P22, SF6 and e34 phages sharing close resemblances in their phage particles appearance. The N-terminal domains of the tailspike proteins (TSPs) of these three phages shares significant similarities, suggesting more structural similarities between them. These phages adsorb to three different hosts: P22 to Salmonella typhimurium, SF6 to Shigella and_e34 to Salmonella newington. However, the e34 TSP has not been as well studied and its 3D structure is not known, unlike the others. We believe an in-depth structural study of the e34 TSP will enhance our knowledge tailspike binding to LPS, and this will in turn offer clues as to genetic engineering TSPs to broaden the host range spectrum of our designed tailspike for both therapeutic and diagnostic purposes. In this research, we made use of a cloning vector called pET30a LIC, which is a highly specific system with faster and high yielding capabilities. The pET30a – e34 TSP has been constructed and confirmed in our laboratory to have our_e34 tail gene of interest and we are currently in the expression and protein characterization stages of the research.

POP09

Peptide-Mediated Inhibition of Staphylococcus aureus

Amber Grace1, Sameer Joshi1, Shree Singh1, Shreekumar Pillai1,*, Komal Vig1, and Balasubramanyam Karanam2

1Center for Nanobiotechnology Research, Alabama State University, Montgomery, Alabama 36104; 2Department of Biology, College of Arts and Sciences, Tuskegee University, Tuskegee, Alabama 36088. *Email: spillai@alasu.edu

Improper use of traditional antibiotics has led to the development and spread of antibiotic resistant bacteria, resulting in difficult to treat infections with increased mortality. The search for alternative novel antimicrobial peptides, to which bacteria have yet to develop resistance, is ongoing. Antimicrobial peptides are naturally occurring oligopeptides of varying length that have antimicrobial activity against a range of microorganisms. In bacterial species, antimicrobial peptides are generally cationic, a-helical, and mainly target gram-positive bacteria. S. aureus is a gram-positive bacterial species that is implicated in instances of wound and medical device infections. Antimicrobial peptides are ideal alternatives to antibiotics to slow the development of antimicrobial resistant bacteria and to avoid increase in mortality. In this study, six novel antimicrobial peptides, SSU2, SSU3, SSU4, SSU6, SSU7, and GM-1, were screened with minimum inhibitory concentration (MIC) assays to determine if any demonstrated antimicrobial effects against S. aureus. SSU (2, 3, 4, 6, and 7) were tested at concentrations of 100 uM, 50 uM, and 25 uM. GM-1 was tested at concentrations of 100 ug/mL, 50 ug/mL, and 25 ug/mL. Since all peptides were dissolved in DMSO, a control with DMSO was included to determine the effect of DMSO on bacterial cell viability. Optical density was measured at 0, 24 and 48 hours to determine microbial inhibition. GM-1 demonstrated the greatest inhibitory potential against S. aureus. DMSO did decrease S. aureus viability in the absence of the antimicrobial peptides.

The properties of GM-1 should be investigated further since it demonstrated the greatest inhibitory potential against S. aureus. While DMSO did have a small effect on the viability of S. aureus, using less DMSO should reduce its effect. Additional peptides that are water and lipid soluble will be tested in future studies as alternatives to DMSO soluble peptides. 

Safdar N and Maki (2002) The commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant Staphylococcus aureus, enterococcus, gram-negative bacilli, Clostridium difficile, and Candida. Ann. Intern. Med., 136, 834-44

Zhang LJ and Gallo RL (2016) Antimicrobial peptides. Curr. Biol., 26, R14-9

Foster T (1996) "Staphylococcus," in Medical Microbiology, S. Baron, Ed., Galveston (TX)

POP10

Polymer Coated and Peptide Functionalized Silver Nanoparticles Inhibit Bacterial Pathogens

D’Andrea Ashmore , Atul A. Chaudhari ,Shree R. Singh, Don R. Owen, Chris Palazzo, Robert D Arnold, Michael E. Miller, Brandi Barlow, Brett Barlow, Talia Harper, Komal Vig, Shreekumar R. Pillai*

Center for Nanobiotechnology Research, Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104. *Email: spillai@alasu.edu

Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica Serovar Typhimurium cause various ailments such septicemia, cellulitis, impetigo and intestinal disease.  Silver nanoparticles (AgNP), silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) are known for their antibacterial activity, and may prove to be beneficial in the medical field.  In this study we compared the antibacterial efficacy of two polymer coated silver nanoparticles either containing 10% Ag, or 99% Ag in relation to plain uncoated silver nanoparticles (AgNP).  Our study also included SWCNTs-Ag that were functionalized covalently with an antimicrobial peptide TP359 (FSWCNTs-Ag). Atomic force microscopy, Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM) were used to characterize the nanoparticles, and their antibacterial efficacy was compared by the minimum inhibitory concentration (MIC) and bacterial growth curve assays, followed by molecular studies using scanning electron microscopy (SEM) and (qRT-PCR).  Both polymer coated nanoparticles inhibited the growth of E. coli at lesser concentration compared to AgNP, showing that the polymer coating stabilized the nanoparticles and made them more effective. The minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (7.8-3.9 µg/ml) compared to plain SWCNTs-Ag (62.6 µg/ml). 

Figure 1. IFUs recovery of C. muridarum from vaginal swab cultures of mice. Mice were challenged with 105 IFUs of live Cm, three-weeks after the last-immunization. Each bar represents the mean IFUs per time-point.

1.  Bryaskova R, Pencheva D, Nikolov S, and Kantardjiev T (2011) Synthesis and comparative study on the antimicrobial activity of hybrid materials based on silver nanoparticles (AgNps) stabilized by polyvinylpyrrolidone (PVP). J Chem Biol., 4:185-91.

2. Chaudhari AA, Jasper SL, Dosunmu E, Miller ME, Arnold RD, Singh SR, and Pillai S (2015) Novel pegylated silver coated carbon nanotubes kill Salmonellabut they are non-toxic to eukaryotic cells. J Nanobiotechnology, 13, 23

POP11

Protective efficacy against Chlamydia intravaginal challenge in BALB/c mice immunized with MOMP encapsulated PLGA 85/15 nanoparticles

Rajnish Sahu, Richa Verma, Saurabh Dixit, Skyla Duncan, Shree R. Singh, and Vida A. Dennis*

Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL 36104. *Email: vdennis@alasu.edu

Chlamydia trachomatis (Ct) remains the leading sexually transmitted bacterial infection worldwide but yet no vaccine to prevent its spread. Attempts to develop vaccines using whole or subunits have failed due to inefficient delivery systems (Dixit et al., 2017; Sahu et al., 2018). We have recently developed a Chlamydia nanovaccine by encapsulating its recombinant major outer membrane protein (rMOMP) (Fairley et al., 2013) in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 nanoparticles (PLGA-rMOMP). In the current study, we assessed the systemic and mucosal antibody responses and the protective efficacy of the PLGA-rMOMP nanovaccine against a live C. muridarum (Cm) challenged infection in immunized mice. Female BALB/c mice were immunized subcutaneously three times at two weeks interval with 50 µg each of either PLGA-rMOMP or bare rMOMP and sacrificed two weeks after the last immunization to measure rMOMP-specific systemic and mucosal antibodies. Serum and cervico-vaginal washes were collected to measure IgG including subtypes IgG2a, IgG2b (Th1) and IgG1 (Th2) responses by ELISA. Immunized mice were challenged intravaginally with 105inclusions forming units (IFUs) of Cm, swabs were collected for three weeks and cultured to quantify bacterial shedding by inoculating McCoy cells. PLGA-rMOMP immunized mice showed enhanced systemic and mucosal IgG antibodies response followed by IgG1 and IgG2b, as compared to bare rMOMP. Moreover, the PLGA-rMOMP immunization induced complete (60% of mice) and partial (40% of mice). Our results show that PLGA-rMOMP stimulates systemic and mucosal antibodies and provides protective efficacy against a Chlamydia infection in mice. This research was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) under Award Number R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) grants. 

Dixit S, Sahu R, Verma R, Duncan S, Giambartolomei GH, Singh SR et al (2017). Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4(+) T cells. Biomaterials 159, 130-145. doi: 10.1016/j.biomaterials.2017.12.019.

Fairley SJ, Singh SR, Yilma AN, Waffo AB, Subbarayan P, Dixit S et al (2013). Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine. Int J Nanomedicine 8, 2085-2099. doi: 10.2147/IJN.S44155.

Sahu R, Verma R, Dixit S, Igietseme JU, Black CM, Duncan S et al (2018). Future of human Chlamydia vaccine: Potential of self-adjuvanting biodegradable nanoparticles as safe vaccine delivery vehicles. Expert Review of Vaccines, doi: 10.1080/14760584.2018.1435279.

POP12

Detection of the Key Hg-methylating Gene in Contaminated Soil from Tennessee

Yazeed Abdelmageed, Carrie Sanders, Timothy Egbo, and Boakai K. Robertson*

Microbiology Program, Department of Biological Science, Alabama State University, Montgomery AL 36104. *Email: brobertson@alasu.edu

Methylmercury (MeHg) is a well-recognized health hazard. It is a pervasive environmental contaminant that accumulates in the food chain, mainly in fish and fish product being the dominant source, and consequently poses a high health risk to humans and animals (Wood 1974). Previous data from the Oak Ridge National Laboratory in Tennessee, show tons of mercury were lost to the environment during the mid of last century contaminating soils, groundwater, air, and the East Fork Polar Creek (EFPC). In nature, MeHg is produced from inorganic mercury predominantly by anaerobic bacteria (Hintelmann 2010). Our measurements of MeHg  in Oak Ridge soil samples revealed elevated levels consistent with the total amount of mercury observed throughout the year, suggesting presence of predicted methylators in that soil.  In this study, we report detection of the key Hg-methylating gene, hgcA, required for microbial mercury methylation in Oak Ridge soil (Parks et al 2013), supported by the soil-DNA sequencing data. Two families, Geobacteraceae and Syntrophobacteraceae dominated other Deltaproteobacteria associated with mercury methylation by 0.1% and 2.1% respectively. Using primers specific for known methylating bacteria, Desulfovibrio desulfuricans ND-132, Geobacter sulfurreducens PCA and Desulfomonile tiedjei DCB-1, four samples were found positive that represent the high  Hg- level in winter and summer soil-cultured samples in selective media and low Hg-level in fall and spring soil-cultured samples. This result was confirmed by sequencing of PCR amplicons and equivalent recovered bands resolved in 1% agarose gel. Six frame translated  sequencing results were used to search for the HgcA putative corrinoid protein -highly conserved motif, N(V/I)WCA(A/G)GK. This results revealed possible MeHg sources in Oak Ridge soil and might contribute to the remediation process.

Wood JM (1974) Science, 183, 1049

Hintelmann H (2010) Met. Ions Life Sci., 7, 365

Parks JM, Johs A, Podar M, Bridou R, Hurt RA, Smith SD, Stephen J. Tomanicek SJ (2013) Science, 339, 1332-1335.

POP13

Recent Advances in Bacteriophage Therapy Highlighting Treatment of MDR Infections

Karthikeya Venkatesan, Joseph Ayariga, Doba Jackson, and Robert Villafane*

Department of Biological Sciences, PhD Program Microbiology, Alabama State University, Montgomery, Alabama 36104. *Email: rvillafane@alasu.edu 

The alarming rise of antibiotic-resistant bacteria is a growing public health concern. The challenges faced by diminished efficacy of current antibiotics, and slow development of new antibiotics has revitalized interest in phage therapy. Since their discovery in 1915, bacterial viruses (or phages) have been known for their ability to infect and lyse specific bacterial hosts. Discovery of potent antibiotics like Penicillin (1928) and Sulfonamides (1932), overshadowed phage usage and research. However, now there is an urgent need to develop novel strategies to combat rising prevalence of multi-drug resistant pathogens (MDRs) especially clinically important bacteria such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, Escherichia coli and Staphylococcus aureus. Promising results have been shown in using phage preparations to control MDRs infections in humans and animals, as well as reduce food contamination. Experimental studies have indicated that phages can tolerate the acidity of stomach, making them useful for the treatment of intestinal colonization of pathogenic bacteria such as E.coli, Salmonella, Campylobacter, and Helicobacter.  Current research is also focusing on applications in using phages in drug delivery systems, vaccine production and nanomedicine. Successful phage administration and treatment relies upon optimizing variables such as delivery routes, monophage/polyphage formulations, dosage and timing post-infection. This talk highlights relevant case studies and recent clinical phage research demonstrating remarkable efficacy in treatment and prognosis of chronic and intractable bacterial infections,  infections where  antibiotic treatments have failed, and those complicated by associating factors like poor circulation, multiple pathogens etc. All of this reinforces the effectiveness of phage-based therapeutic applications.

POP14

High Resolution 3D Printing of a Polymeric Bio-scaffold

Hianxiao Huang and Derrick Dean*

Biomedical Engineering Program and Integrated Bioengineering and Advanced Materials Center, Alabama State University, Montgomery, Alabama 36104. *Email: ddean@alasu.edu

Additive manufacturing, commonly known as 3-D printing, is rapidly gaining acceptance as a viable approach for fabrication of tissue scaffolds with precise control of architecture, composition and properties.  Further studies are needed to advance this technology.  In this study, we printed porous 3 dimensional polymeric(polycaprolactone) tissue scaffold using a 3-D bioplotter.  The pore size was varied from 192 to 420 _m.   The corresponding porosity was characterized and correlated with the morphology.  The bioinks used for printing were optimized with the appropriate viscosity for high resolution printing.  Rheological studies demonstrate a shear thinning behavior of the ink which is a prerequisite for printing. Finally, the tensile tests of 3D printed dog bone sample demonstrated an excellent mechanical property compared to other common polymeric biomaterials. We believe this work will broaden our understanding to the 3D printing at a higher resolution and finer pore size, and specifically the fabrication of cellulose acetate scaffolds for tissue engineering.

POP15

Usefulness of Natural Antimicrobials to Combat Pathogenic Aeromonas spp.

Daphne Topps, Boakai K. Robertson, and Mamie T. Coats*

Department of Biological Sciences, Alabama State University, Montgomery, Alabama 36104, *Email: mcoats@alasu.edu

Disease outbreaks in aquaculture that are caused by pathogenic bacteria have an enormous impact with losses of billions of dollars worldwide.  Pathogenic Aeromonas species are responsible for the majority of diseases in aquaculture. Unfortunately, Aeromonas spp. have developed multidrug resistance to several classes of antibiotics due to antibiotic overuse.  Phytochemicals which are bioactive, non-nutrient plant compounds are being proposed as an effective and eco-friendly alternative to antibiotics. Our goal was to determine the efficacy of phytochemicals in inhibiting Aeromonas growth.  Aeromonas hydrophila, Aermonas caviae, and Aeromonas veronii were grown in an overnight culture containing a given phytochemical.  The resultant bacteria were plated on Muller-Hinton media and incubated overnight at 29°C.  Black pepper and ginger extract inhibited survival at 25% (v/v) concentration. Dillweed oil and cumin seed oil inhibited the growth in a 10% (v/v) solution. Clove bud oil, lemon oil, furfural, and coriander inhibited the growth of the Aeromonas species in 1% (v/v) solutions. Future studies will further identify and examine the active antimicrobial agent in the most effective phytochemicals.

POP16

Neuroligin 4x, a Neural Cell Adhesion Molecule, in Breast Cancer

Henry J. Henderson1, Balasubramanyam Karanam2, Rajeev Samant3, Komal Vig4, Shree R. Singh4, Clayton Yates2, and Deepa Bedi1,*

1Department of Biomedical Sciences, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama 36088; 2Center for Cancer Research, Tuskegee University, Tuskegee, Alabama 36088; 3Department of Pathobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 4Center for Nanobiotechnology Research, Alabama State University, Montgomery, Alabama 36104. *Email: dbedi@tuskegee.edu

Neuroligins are neural cell adhesion molecules that are implicated in heterotopic cell adhesion. Our previous studies using a selection from a combinatorial random peptide library against breast and pancreatic cancer cell lines identified several peptides mimicking neuroligin (NLGN-1, 3 and NLGN4X respectively). In this study, we investigated the expression, relevance and functional significance of Neuroligin 4X in human breast cancer. The correlation between NLGN4X levels and clinicopathologic parameters were established within Oncomine datasets. As a proof-of- principle, we evaluated survival by generating Kaplan–Meier plots using publicly available microarray datasets. To observe the effect of NLGN4X gene knockdown, MDA-MB-231 cells were transfected with NLGN4X-specific siRNA. Post transfection, wound healing and cell viability assays were performed to determine the effect of NLGN4X knockdown on migration and proliferation. Apoptotic outcomes were examined through detection of caspase activation and Annexin V-FITC methods by using flow cytometry. NLGN4X showed abundant expression in breast cancer tissues. The evaluation of bioinfomatic datasets revealed that NLGN4X expression was higher in triple negative breast cancer (TNBC) and in metastatic tissues. Interestingly, high NLGN4X expression correlated with a decrease in relapse free-survival in TNBC. RT-PCR, flow cytometry and immunofluorescence validated that NLGN4X expression was high in MDA-MB-231 as well as MCF-7 TGF_, which suggests that NLGN4X is associated with the mesenchymal phenotype. Knockdown of NLGN4X expression by siRNA significantly decreased cell proliferation(P<0.001) and migration(P<0.05) in MDA-MB-231 in conjunction with the induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry(Figure.1).  Our findings suggest that NLGN4X could represent a novel biomarker and therapeutic target. This is the first study to link the expression of neuronal cell adhesion molecules, neuroligins, to breast cancer. We intend to continue the investigation of the role of NLGN4X to elucidate the mechanistic role of this adhesion protein in breast cancer progression and metastasis.

Figure 1. Knockdown of NLGN4x by gene specific siRNA resulted in elevated levels of apoptotic markers (A) and the induction of apoptosis (B).

Bedi D, Gillespie JW, Petrenko VA. Selection of pancreatic cancer cell-binding landscape phages and their use in development of anticancer nanomedicines. Protein Eng Des Sel. 2014; 27(7):235-243.

Samarelli AV, Riccitelli E, Bizzozero L, Silveira TN, Seano G, Pergolizzi M, et al. Neuroligin 1 induces blood vessel maturation by cooperating with the alpha6 integrin. J Biol Chem. 2014; 289(28):19466-19476.

Wai Wong C, Dye DE, Coombe DR. The role of immunoglobulin superfamily cell adhesion molecules in cancer metastasis. Int J Cell Bio. 2012;340296.

POP17

Analyzing the Effects of Antimicrobial Layer-by-Layer (LbL) Films under Various Conditions

Stephanie Barrow1, Brielle Weiner2, Catherine Picart3,4, Daeyeon Lee2, B. K. Robertson1,*,

and Mamie Coats1, *

1Microbiology Doctoral Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL; 2Department of Chemical and Bimolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania; 3CNRS, UMR 5628, LMGP, F-38016, Grenoble, France; 4Institut National Polytechnique de Grenoble, University Grenoble Alpes, F-38016 Grenoble, France. *Email: brobertson@alasu.edu, mcoats@alasu.edu

The transmission of infection is increased after natural disasters due to poor sanitation and other conditions. Exposure to poor sanitation can lead to the risk of individuals being infected by pathogenic agents. Our solution is to use antimicrobial smart coatings to prevent the transmission of infection. The overall goal is to test and optimize layer-by-layer (LbL) films that will release antimicrobials over long periods of time. The chitosan (CHI)/poly-L-glutamic acid (PGA) films were built by the TECAN Freedom EVO 100 deposition robot or manually. The reproducibility of the manually built films was analyzed by using the TECAN Infinite 1000 fluorescent spectrometer. The surface characterization and/or homogeneity of the films were analyzed by using the LSM700 ZEISS confocal microscope. Bocillin FL, a derivative of penicillin, was the antibiotic that was loaded in the films.  The loading and release kinetics of bocillin were measure by using the TECAN Infinite 1000 fluorescent spectrometer. The TECAN Infinite 1000 fluorescent spectrometer showed the linear growth and slight exponential growth of the manually built films when different pH conditions of the polyelectrolytes were used. The LSM700 ZEISS confocal microscope showed the homogeneity of the films that were manually built at pH 5. No significant differences were found between the various loading and release conditions.There were no significant changes detected when the various release pH conditions were used for the release of bocillin FL. More conditions of the polyelectrolytes should be tested to determine the conditions that are needed for exponential growth. Future work includes testing different antimicrobial LbL systems (linear versus exponential).