MACUB (2021) Conference

Abstract:

The impact of climate change is being felt worldwide: global temperature is rising, snow covers are decreasing, glaciers are retreating, ice sheets are shrinking, sea levels are rising, extreme weather events are more frequent, and oceans are warming and acidifying. Humanity is facing a big environmental challenge which not only impacts our habitat and life of all other organisms on our planet but will also have ramifications on our health. In our research study, we have conducted a detailed examination of the scientific evidence proving the relationship between climate change and various fatal human diseases. Our findings indicate that variations in climatic conditions, such as temperature, rainfall patterns, and humidity, can increase the incidence of respiratory infectious disease- tuberculosis, and alter the incidence of waterborne diseases, especially diarrheal diseases. Also, an increase in the longevity of mosquitos and the development of malaria parasite, and consequently, transmission of malaria has been noted. Climate change could alter the dispersion of primary pollutants and secondary pollutants such as ozone gas. These pollutant gases have been linked to increased incidence of congestive heart failure and acute myocardial infarction. Furthermore, the incidence of allergic diseases and asthma are also likely to increase with climate change. From our studies, we conclude that identifying emerging disease risks is crucial to assess our vulnerability, and to determine specific areas where public health efforts are required.   

Abstract: COVID-19 is spread primarily by respiratory droplets discharged into the air by coughing, sneezing or talking. In the spring of 2020, governments around the world recommended or mandated that the general public wear face masks as a barrier to droplet transmission. It has been known for some time that surgical masks are a potential source of bacterial shedding in hospitals. Recently, news media reported the detection of bacterial pathogens on six masks worn by elementary school children in Florida. Evidence indicates that bacteria on the external surface of a mask are likely from the wearer. Disposable face masks are frequently reused increasing the chance for contact transmission between hands and mask. This pilot study focused on isolating and characterizing bacteria and fungi on disposable face masks and assessing the relationship between mask reuse and contamination. Twenty-three used disposable masks were donated anonymously. The three-ply, non-woven, polypropylene masks were placed in sterile containers of Lauria broth for 20 minutes. The spread plate procedure was used to assess microbial contamination. Bacteria were cultivated on Lauria broth agar plates incubated at 37oC for 48 hours to estimate bacteria levels (cfu/ml/piece). Isolated bacteria were Gram-stained and selected colonies were identified with 16S rRNA gene sequence analysis. All six isolates are potentially pathogenic including Staphylococcus aureus and five Enterobacteriaceae: Phytobacteria, Klebsiella pneumonia, Salmonella Thompson, Klebsiella oxytoca, and Pluralibacter geroviae. Lauria broth from several masks was inoculated on 3M yeast fungi (YF) Petrifilm and incubated at room temperature for 5 days for fungal counts (cfu/ml/piece). Preliminary results indicate that mask reuse increases contamination. Future work will include 16S rRNA identification of the remaining bacterial isolates as well as the processing of additional face masks. Establishing a direct link between reuse and contamination could lead to educational campaigns designed to increase hand-mouth cleanliness and decrease the reuse of disposable masks.

Abstract: Plants lack an adaptive immune system and instead rely on an innate immune system that allows them to defend themselves against microbial pathogens. Some of these pathogens include bacteria, viruses, fungi, oomycetes and nematodes. Pathogens are either very host specific or can affect a wide arrange of plants. These pathogens enter through wounds, natural openings, vectors, stylets or mechanical pressures. Once in the host, pathogens then use molecular strategies to cause disease to the plant and begin colonization. Our research focus is on oomycetes as a plant pathogen. Oomycetes use sophisticated molecular strategies called effector proteins to sabotage the defenses of their hosts. These proteins, often manipulate signaling pathways in the plant in order to cause disease. Various plant hormones such as salicylic acid, jasmonic acid and ethylene trigger these signaling pathways and are the target of pathogen effectors. These pathways induce several immune responses in the host plant such as those inhibiting pathogen proliferation (PAMP-triggered Immunity or PTI) and those recognizing the effector right away (Effector-triggered immunity or ETI). Each year, agronomically important crops such as maize, wheat, rice and potatoes are lost due to pathogen attack, which not only cost the agricultural industry billions, but plays an effect on world hunger. Throughout this research, I used the Brugmansia to model how infection would arise in Arabidopsis thaliana to study plant-microbial interactions. The aim is to understand and possibly figure out how to avoid pathogen infection due to the action of effector proteins. Phytophthora sojae is an oomycete that causes stem and root rot of soybean. In this research, we aim to show that effectors from P. sojae act as suppressors of defense gene pathways in plants such as Arabidopsis. Using bioinformatics information, a virtual simulation and in-planta experiments, we have reached a better understanding of how effectors target the innate immunity that plants harbor.

Abstract: Cervical cancer is a type of cancer that affects the region of the women’s reproductive system called the cervix. This cancer attacks two different cells the glandular cells and the squamous epithelial cells but starts as Cervical intraepithelial neoplasia by healthy cervical cells going onto dysplasia. According to the American Cancer Society, the five-year survival of this neoplasm is 92%, 58%, and 17% for localized, regional, and distant diseases. To improve the prognosis of this cancer, one line of study is to identify novel compounds with strong anti-cancer effects. Here, we studied the effects of diminazene aceturate (DIZE) on HeLa cells, a human cervical carcinoma cell line, after treatment for 48 hours. The cytotoxic assays we used include acridine orange-ethidium bromide (AO-EtBr) live-dead staining, casp3-7-specific staining, and JC-10staining for detecting changes in mitochondrial membrane potential. Cell count of images from AO-EtBr assay revealed a greater percentage of dead cells associated with 100 uM DIZE (37.8% ±3.03%) compared to DMSO (Dimethyl Sulfoxide, solvent used to dissolve DIZE) -- (9.1% ±0.91%) (p<<0.05). In the AO-EtBr assay, AO which is membrane-permeable, stains live cells green, and EtBr which is membrane-permeable stains dead cells red (EtBr+). A similar counting procedure was carried for cells stained with a caspase3/7-specific dye, (Cell Event caspase3/7 green reagent). Caspase signaling consists of a series of enzymes that are involved in programmed cell death. In this assay, the cell count of images revealed a greater percentage of cells that stained green (indicating caspase 3/7 activation) with the dye at treatment with 100 uM DIZE (40.94% ± 1.05%) as compared to DMSO-- (6.67% ± 4.13%) (p<<0.05). We also study the effect of DIZE on mitochondrial membrane potential using the cationic dye, JC-10. The mitochondrion is a cellular organelle that is important for capturing energy from nutrients. A double-membrane structure, the mitochondria is a charged organelle (charge difference between the inside and outside of the mitochondrial membrane). Disruption of this charge can lead to cell death. With the JC-10 dye, the loss of mitochondrial membrane potential is followed by a red-to-green shift. Therefore, microscopically, live cells are stained with JC-10 dye contain red puncta or aggregates which are absent from or diminished in dead cells, which stain predominantly green with the dye. In our study, JC-10-stained cells treated with 100 uM DIZE were predominantly green while those treated with DMSO stained orange to red. Our results suggest that diminazene aceturate causes cytotoxicity via caspase activation and loss of mitochondrial membrane potential, in human cervical carcinoma.

Abstract:

Group B Streptococcus is the number one cause of neonatal mortality due to infections such as pneumonia, bacteremia and meningitis, by passing from mother during birth. The preferred treatment for GBS is Penicillin G, however there can be treatment delay if the bacteria strain is penicillin tolerant. When a GBS strain is tolerant it can temporarily withstand, which means that the bacteria can regrow once again after the penicillin treatment. This experiment is conducted to determine a specific gene that is related to penicillin tolerance and create an assay to develop a primer that can be utilized during PCR testing.

The first trial is to confirm tolerance using a macrobroth dilution for penicillin killing and a regrowth tolerance assay. In a 96-well plate, susceptible strain, A909 and the tolerant strain, O90R will be incubated and grown overnight before it is treated with Penicillin G at different concentrations. The plate is again incubated overnight, and treated with Penase, an enzyme- based product that inactivates Penicillin G. After a third incubation period, the final results are measured on a spectrophotometer. For the second trial, Both strains were stained with SYTO9 and PI to measure its cell viability, membrane permeability and damage. The plate was incubated for 10 minutes in the dark, then SYTO9 and PI were excited at 444 nm laser, and were detected at 538 and 612nm.

After the penicillin treatment,there was a decrease of cells for the O90R strain, and the A909 strain. After the Penase U treatment, O90R cells increased for all penicillin concentration. For A909, there was no growth for the cells with high concentration of penicillin. For the cells with low concentration of penicillin, there was some growth. The A909 strain showed a tendency of declining SYTO9 concentration as penicillin G increased and after penase treatment. The O90R strain showed an increase of SYTO9 after penase treatment. So it was concluded that the O90R strain is tolerant strain, therefore it did not get lysed during the penicillin treatment and had regrown after the penicillin was deactivated. Whereas the A909 strain was lysed during the penicillin treatment and was unable to regrow when the penicillin was deactivated.      

Abstract: The Food and Agriculture Organization of the United Nations estimates that between 20 and 40 percent of crop production is lost to pests worldwide each year. Plant diseases cost the global economy approximately $220 billion in crop loses annually. This blight on crops has led to a massive decrease in food supply allowing 800 million people to suffer from hunger and starvation. Pathogens and pests are the major cause of such crop losses and account for many issues that we see in plants. Bacteria, fungi, nematodes, and oomycetes are examples of different types of pathogens that lead to such high losses. These pathogens use sophisticated molecular strategies called effector proteins to sabotage the defenses of their host. Effectors from these pathogens are secreted into the interior of plant’s cells, corrupting specific organelles such as the mitochondria, nucleus, cytoplasm, and even chloroplasts to suppress host defense and cause disease. In defense of this onslaught, plants preserve their health via their immunity response, which has been found to be a cascade of events and responses. The first phase is called Pathogen-Associated Molecular Pattern Triggered Immunity (PTI) where the plant triggers a primary immune response such as releasing reactive oxygen species to stop the pathogen. If the pathogen evades this phase, it can secrete its effectors leading to Effector Triggered Susceptibility (ETS) causing the plant to become compromised or diseased. The final phase is called Effector Triggered Immunity (ETI) where the effectors are recognized by plant resistance proteins (R Proteins) leading to cell death in the affected area. Our research focuses on effectors and their effect on plant defenses. In order to understand how the pathogen effectors interact with host defense for the purpose of causing disease, we designed and executed an experiment that would allow us to test our theories. We hypothesized that effectors from oomycete plant pathogens would interfere with the expression of host defense genes that are important in plant defense hormonal pathways such as jasmonic acid and salicylic acid. We virtually tested this theory using a simulation-based software, Plant Simulation Laboratory where we identified genes that were most critical in the host. We then confirmed our virtual results through in planta experiments where we performed qRT-PCR to determine whether the effector proteins where able to suppress the host defense genes in pathogen-treated plants. Our results show that oomycete effectors Avh73 and RxL23 were unable to target the defense genes in question and did not suppress their expression. We are currently testing other host defense genes that may be the targets of these effectors in planta.

Abstract: Curcumin, derived from the plants of the Curcuma longa species, more popularly known as turmeric. Prior studies reported that curcumin possesses antimicrobial, anti-inflammatory, antioxidant, and antiparasitic activities. F2, a formulation containing patented lipid-soluble green tea polyphenols, was also included in this study to test its antimicrobial effect. The bacteria used in this study were Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). E. coli, a Gram-negative bacterium, can cause various bacterial infections including urinary tract infections (UTIs), pneumonia, and cholecystitis. B. subtilis, a Gram-positive pathogen, is known to cause pneumonia, endocarditis, and bacteremia. This study aims to evaluate the antimicrobial effect of curcumin, F2, and the combination of both compounds against B. subtilis and E. coli with various assays including microplate, colony-forming unit (CFU), and disk diffusion assays. The growth analysis results indicated that the minimum inhibitory concentrations (MICs) for curcumin and F2 on B. subtilis and E. coli are 100 µg/mL and 10%, respectively. The CFU data showed growth inhibition ranging from 42.86% to 95.29% against B. subtilis and 87.17% to 99.93% against E. coli after treating with curcumin. As for F2, the CFU data showed inhibition ranging from 68.97% to 98.28% against B. subtilis and 98.22% to 99.88% against E. coli after treatment. A time-course (1, 5, 10, and 30-min) and study was carried out to determine the antibacterial efficacy of F2. The results indicated that F2 can reach 2.32 and 2.53 log reduction in E. coli and B. subtilis, respectively, suggesting that the lipid-soluble green tea polyphenol-containing formation could be a natural alternative bactericidal agent.

Abstract: Over the years biofilm formation has become one of the leading causes of infections in hospitals due to their resistance to antibiotic treatments as well as the immune system. There has been a dire need for new compounds that possess antimicrobial properties that not only inhibit the growth of biofilm but also potentially kill pathogens. As a result, many scientists have turned to natural compounds as a possible solution to the ongoing concern. In this research, our goal was to determine the antimicrobial and antibiofilm activity of two amino acids, N-acetylcysteine, and L-histidine, on pathogenic bacteria and fungi. To achieve this goal, we tested the antibiofilm and antimicrobial effect of the two amino acids against 14 different strains of bacteria and 3 strains of fungi using a biofilm and broth assay. Results showed that N-acetylcysteine had a strong bactericidal effect against all the tested pathogenic bacteria at 8mg/ml. N-acetylcysteine was also capable of biofilm inhibition in some bacteria. L-histidine demonstrated minor inhibition of biofilm formation activity against Staphylococcus aureus and Escherichia coli, and no bactericidal activity against all tested pathogens. Future studies will focus on studying the cytotoxic effects of amino acids on human cells.