Summer Undergraduate Biomedical Researcher Programs
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|1. "Testosterone and obesity-associated asthma" - Sathish Venkatachalem, Associate Professor, Pharmaceutical Sciences|
Obesity is associated significantly with the development of asthma, worsening asthma symptoms including increased airway hyperresponsiveness (AHR) and remodeling, and poor asthma control. The prevalence of asthma is increased in obesity, particularly in adult women. In the United States, asthma is significantly more prevalent in patients with obesity compared with the remainder of the population (14.6% in obese vs. 7.9 % in normal females, 7.1% in obese vs. 6.1% in males). The major biological factor that could be changed is the physiological levels of sex hormones, especially estrogen and testosterone. Previous reports show that estrogen and testosterone have a crucial role in regulating airway inflammation and remodeling during asthma occurrence. In asthma pathophysiology, airway smooth muscle (ASM) plays a critical role in contributing to both exaggerated airway narrowing as well as remodeling. However, the influence of obesity on ASM functions is still unexplored. The association between asthma and obesity is complex and multifaceted and may be regulated by many different mechanisms. In obesity, leptin which is an important obesity-related hormone contributes to chronic systemic inflammation and cell proliferation, which are relevant to asthma pathogenesis. With the above consideration, we believe a strong association between the anti-inflammatory effect of testosterone and leptin signaling in the pathophysiology of obesity-associated asthma. In this context, our overall hypothesis is that testosterone regulates the expression and function of leptin in ASM and regulates inflammation, AHR, and remodeling. Additionally, with this proposal, we aim to provide research experience and training to an undergraduate student at NDSU. The student candidate will perform and/or assist many of the basic biomedical science lab duties as well as be trained on several cutting-edge techniques, this will set the stage for a student’s future towards biomedical-based opportunities.
|2. "Testing the efficacy of a novel antiproliferative mechanisms in cancer" - Sijo Mathew, Assistant Professor, Pharmaceutical Sciences|
Increased proliferation and migrations are some of the major features present in cancer cells compared to normal cells. There are many factors contributing to cancer cell proliferation including the signaling from tumor microenvironment that is transduced to the cytoplasm and actin cytoskeleton through cell surface receptors. Recently our laboratory discovered a novel strategy to modify the functions of one of the major receptors, integrin β1, to turn on the anti-proliferative pathways in cells. This proposal is investigating the efficacy of this newly discovered strategy to prevent the progression of cancer using pancreatic and renal cancer models. After evaluating the integrin β1 and associated transporter proteins expression, the role of these proteins in tumor progression will be investigated using genetic modifications in renal cancer (caki-1, caki-2) and pancreatic cancer (PANC-1) cells. 2D and 3D cell culture model will be used to investigate the role of these proteins in tumor cell proliferation.
3. "Impact of Western Diet on the Intestinal Stem Cells in a Diversity-Outbred Mouse Model" - Yagna Jarajapu, Associate Professor, Pharmaceutical Sciences
|4. "Multi-pronged Catalytic Methods for the Introduction of Fluorinated Moieties to Organic Small Molecules" - Hariharaputhiran Subramanian, Research Assistant Professor, Chemistry and Biochemistry|
The basic synthetic organic chemistry is multi-faceted and intimately involved in several aspects of biomedical science. It plays an important role in the development of therapeutics to treat a variety of human diseases. The significance of fluorine containing small organic molecules in pharmaceutical sciences, agrochemistry, materials sciences and biological sciences has grown over the past decade owing to the unique properties of fluorine atom. Despite the profound impact of these compounds, reactions focusing on introducing fluorinated moieties seem to remain elusive in most introductory organic chemistry course curricula to date. Conjugate addition reaction or Michael type reaction is one of the most common and powerful transformation, taught in elementary undergraduate organic chemistry curriculum all over the world. Therefore, introduction of an elusive concept of introducing fluorinated groups using a familiar reaction imply a desired approach to an undergraduate student. For the summer undergraduate biomedical research program, the prospective undergraduate student will work on a project that involves conjugate addition of fluoroalkyl moieties to activated alkenes. This project involves a multipronged catalytic approach to achieve the desired goal. The trifluoromethyl functionalized organic compounds synthesized in this project are basic building blocks for functionalized biologically active compounds. The following scheme shows general description of the method that will be investigated during the 10-week summer period. The student involved in this project will work safely in a group setting and learn organic synthetic techniques. He/she will also learn analysis skills, use of state-of-the-art spectroscopic instruments, and enhance their oral and written communication skills. The student will be a participant in weekly group meetings and social activities. The student will also have opportunities for peer interactions with NSFREU students and NDSU undergraduates.
|5. "Protein phase separation and phase transition in heart diseases" - Ang Guo, Assistant Professor, Pharmaceutical Sciences|
Heart diseases remain the leading cause of death in the United States. Liquid-liquid phase separation of proteins and aberrant phase transition into amyloid-like aggregates play critical roles in cardiac pathophysiology. This summer student project is branched from an ongoing project studying the phase transition of a heart contraction regulator protein. The student will study the effect of a mutation on the phase separation of that protein, as well the cardiomyocyte phenotypes associated with that mutation. Depending on the background and skills of the student, the study can adopt experimental approaches (biochemistry, cellular molecular biology) or in silico approaches (using computational tools to simulate the molecule dynamics). The duties of the student may include: routine bench work of biochemistry and cellular molecular biology; taking care of lab animals; use computational tools to model structure of protein complexes; apply patch clamp technique to examine cardiomyocyte electrophysiology. A pre-knowledge in Linux system and programming is desirable and will help solving technical problems in the project. The skills gained in this project can be applied to a wide array of biomedical research.
|6. "Microfluidic Technology for Isolating Biomarkers" - Yongki Choi, Associate Professor, Physics|
Lab-on-a-chip (LOC) technology offers new possibilities for portable and point-of-care diagnostics of various human diseases. In particular, microfluidic-based LOC methods provide high-throughput, multiplexed, and comprehensive detection of disease-associated protein biomarkers from complex biological samples, greatly simplifying the detection processes using minimal sample volume. For example, liquid biopsies using the microfluidic method have shown tremendous potential for early-stage disease diagnosis, screening, and monitoring. However, obtaining high-quality biomarkers from whole blood in a microfluidic system is technically challenging. Recent advancements in microfluidic technology have led to the development of the hydrodynamic deterministic lateral displacement (DLD) principle for particle separation and fractionation. DLD utilizes a periodic array of obstacles and the asymmetry of laminar flow to separate particles based on size. The DLD process sorts particles by their diameter (Dp) and critical diameter (Dc): particles with Dp greater than Dc will be displaced laterally across the array in a bumping mode, while those with Dp less than Dc will not be displaced laterally and instead follow a zigzag path. The goal of this project is to develop miniaturized, DLD-based microfluidic devices that could be a solution for clinically reliable biomarker separation from unprocessed whole blood for point-of-care blood testing applications. Initially, we will design various DLD structures and fabricate microfluidic devices using a 3D printer and/or optical lithography methods. In parallel, we will test the design using finite-element computer simulation software, from which we will be able to predict particle motions in the devices. Next, we will determine the particle sorting efficiency by testing the devices with polystyrene particles and examining their trajectories. We will be able to identify the important structural parameters (channel dimension, post density, post shapes, etc.) and control parameters (particle concentration, flow rates, separation time, efficiency, etc.) of the device. Also, we will examine potential clogging and jamming problems, as well as slow flow or random diffusion issues in the device.
|7. "Impact of Peroxisome Proliferator Activated Receptor (PPAR) alpha on mitochondria in cardiomyocytes" - Natasha Fillmore, Assistant Professor, Pharmaceutical Sciences|
The proposed research focuses on how diabetes can cause deleterious effects in the heart. Specifically, this project will focus on energy metabolism is regulated in the heart within the context of diabetes. The expected research outcome during this summer is to determine the impact of PPAR alpha (a key transcription factor that increases in the diabetic heart and in known to regulate energy metabolism in the heart) on mitochondria (powerhouses of the cell) in heart cells. This will include measuring the level of key mitochondrial proteins under conditions where PPAR alpha activity has been modulated in heart cells. This research outcome is one of several expected beneficial outcomes for this research experience. A few of these outcomes include: 1) learning technical skills related to laboratory techniques, 2) gaining scientific/critical thinking skills, 3) gaining experience compiling and sharing data in lab meetings.
|8. "The effect of lead accumulation on the behavior, health, and fitness of a wild songbird" - Britt Heidinger, Associate Professor, Biological Sciences; Timothy Greives, Associate Professor, Biological Sciences; Samuel Lane, Biological Sciences|
Despite the U.S. placing a ban on leaded paints and gasoline in the late 1970’s, accumulated stores still exist across North America. Exposure to lead during development has been shown to alter adult behavior, increasing aggression, and decreasing sociality. Urban adult animals, especially birds, are more aggressive than their rural counterparts. Developmental lead exposure is also linked to adult aggression in humans and laboratory animals. However, it is unclear if developmental lead exposure is a key factor influencing greater adult aggression in urban populations. In Fargo, ND, lead paint accumulation is highly variable. Some sections of the city have very high levels categorized within the top 95th - 100th percentile of lead paint accumulation nationally, while other areas of the city have negligible levels. This variation in lead accumulation across a small geographic area provides a novel opportunity to study the effects of lead exposure on a single population of wild songbirds, and test if the levels predicted by the EPA’s Environmental Justice Screening (EJScreen) tool reflect biological accumulation of lead which can have devastating effects on humans, wildlife, and ecosystems. We propose to quantify variation in blood lead levels in adult and nestling house sparrows (Passer domesticus) living in and around Fargo ND, and relate these levels to behavior, immune health, and reproductive success. Specifically, we predict bloodlead levels to be highest in areas classified in the 95th – 100th percentile of lead paint accumulation, and to decrease along the described gradient. Additionally, we predict that in the highest percentile areas, blood lead levels will be negatively correlated with reproductive success (fledging success, nestling mass at fledge) and measures of immune health and cellular health (telomere length, H/L ratios, and Bactria Killing Ability). Finally, we predict that blood lead levels will be positively correlated with adult aggression and negatively corelated with sociality. Undergraduate researchers (REUs) will conduct field work in collaboration with the Timothy Greives and Britt Heidinger lab research teams throughout the breeding season (May-August) and will receive tiered mentoring from Timothy Greives, Britt Heidinger and the post-doc, Samuel Lane. The REU student will gain experience in many techniques including: nest searching, behavioral assays, capturing, handing, blood sampling and processing, and measuring adult and nestling house sparrows. This REU will provide the student with first-hand experience in all aspects of the scientific process, including: study design, sample collection, data analysis, and disseminating the results to the greater community via a presentation and manuscript.
|9. "Artificial Intelligence-Based Patient Identity Verification Using Common Medical Sensors" - Jeremy Straub, Assistant Professor, Computer Science|
Typical biometric sensors, which are used to verify identity, include fingerprint and hand geometry scanners and face, iris and speech recognition sensors. However, many patents interact with numerous other sensors during the course of a physician office or hospital visit. These sensors may be equally useful at identifying patients to prevent identity mix-ups which can lead to incorrect procedures being performed or medications being administered. This project will assess the efficacy of other common biomedical sensors for identity determination. Examples of sensor types that will be assessed include temperature probes, oximeters, glucometers, electrocardiograms, electroencephalograms, electromyograms and respiration rate, heart rate, airflow and pressure sensors. What the Student Researcher Will Do: The undergraduate researcher will build on existing research within the faculty mentor’s lab that has developed a system for identity verification from sensor inputs and develop software that will run on a microcontroller or microcomputer to collect data from multiple sensors. She/he will then collect data from human subjects using this sensing system which will be analyzed to assess the extent to which sensors and sensor combinations can be used to establish subject identity (i.e., differentiate between different subjects). This data will be supplied to an artificial intelligence / machine learning system. The level of accuracy of this identity verification technique will be compared to the performance of traditional identity biometric sensors (based on published results for these types of sensors) and prior results using a public dataset. The undergraduate researcher will prepare a technical paper summarizing the study and results.
|10. "Predicting Drug-like and Toxicological Properties of Nanomaterials: Application of Machine Learning and Computational Approaches" - Bakhtiyor Rasulev, Associate Professor,Coatings & Polymeric Materials|
The discovery of new materials with enhanced properties is one of the main goals in science, but most of this discovery has been done by using trial and error methods, which is inefficient. As the experimental evaluation of materials properties are time-consuming, and in some cases - very expensive, computational approaches are good alternative. The research in Rasulev group is focused on development of predictive models to investigate and design novel bio-based polymeric and nanomaterials, by predicting biological activity properties, toxicity, solubility, degradation, etc. The group applies artificial intelligence (AI), machine learning (ML), computational and cheminformatics methods. Various nanomaterials, including nano-size organic compounds, such as fullerene and its derivatives (FDs), cyclodextrins (CDs) and their complexes are widely applied in materials science, pharmaceutical industry, and (bio) medicine. This research will be focused on the study of these materials in terms of their potential drug-like activity and inhibitory effect on therapeutic targets associated with various diseases, including diabetic disease. Therapeutical drug compounds when enter the biological system usually inevitably encounter and interact with vast variety of biomolecules that responsible for many different functions in organisms. Protein biomolecules (receptors) are the most important functional components and will be used in this study as target structures. Receptor-nanomaterial binding activity data will be investigated in this study by using various AI, machine learning and cheminformatics methods, including artificial neural network (ANN) algorithms. The Quantitative Structure Activity Relationships (QSARs) models for prediction of anti-diabetic activity and toxicity will be developed. All obtained data can provide important information for the further potential use of investigated nanomaterials as promising medicinal agents.
|11. "Bacterial Protein Function Prediction Using Machine Learning" - Harun Pirim, Assistant Professor, Industrial & Manufacturing Engineering|
The project aims to reveal protein function information from bacterial genome using publicly available data sets and databases. Data analytics and machine learning will be employed. Expected outcomes of the project are development of the best classifier, hands-on expertise on biomedical data analytics tasks, and a draft conference paper to be submitted to a biomedical conference. Proteins are essential for the functioning of living organisms. As the number of proteins being discovered grows, the characterization of their functions lags behind due to experimental limitations among others. Therefore, using computational methods to predict protein function is critical to support biological experiments. Proteins have physicochemical, sequence-based properties, interactions with other proteins. These properties are usually stored in databases for researchers to unite and explore new information on specific problems. The information in these databases can be used in unique machine learning pipelines to predict functions of proteins. Besides, deep learning approaches using representation learning avoid feature engineering employed in classical machine learning approaches to predict functions. Predicting protein function involves assigning it to a specific category, and this can be approached as a classification problem. These assignments often take the form of Gene Ontology (GO) term assignments. Instead of predicting GO terms, some predictors identify connections between genes that share similar functions, by outputting a network where genes are represented as nodes and an edge between two genes signifies that they share a common function. Different types of classifier models can be employed to achieve prediction, by utilizing supervised, unsupervised or semi-supervised learning techniques. While deep learning techniques have become popular for identifying relevant features and building accurate predictors, traditional machine learning methods like logistic regression have been found to be more effective in some cases. The project investigates a unique data analytics and machine learning approach using existing tools, comparing machine learning models with hyper parameter optimization. This research contributes biomedical research skills and knowledge creation. The tasks to complete include:
|12. "Asymmetric Synthesis, Structure-Activity Relationship and Biological Evaluation of Chiral Stenocarpoquinone A and derivatives" - |
Roberto Gomes, Assistant Professor, Pharmaceutical Sciences
|13. "Investigating the role of platelets during sepsis" - Elisabetta Liverani, Assistant Professor, Pharmaceutical Sciences|
Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high morbidity and mortality. Current sepsis treatments are limited to supportive therapies, and specific pharmacologic treatments that could greatly improve patient outcomes have not yet been developed. Sex differences in the morbidity and mortality of sepsis have been highly observed in animal models and human diseases. To date, females have shown decreased mortality and organ failure in mice and humans compared to their male counterparts. However, the lack of studies comparing both genders limits our capacity to evaluate the extent of sex-related differences. Platelets are important cells during sepsis, in particular for their interaction with other immune cells. There are also sex-related differences in how platelet respond to activation. Purinergic signaling represents a novel regulatory mechanism in immune cell physiology. Cells respond to activation with the release of cellular ATP, which regulates cell functions. In sepsis large amounts of ADP released by tissue damage disrupt these regulatory purinergic signaling mechanisms, leading to immune dysfunction that promotes the pathophysiologic process involved in sepsis. Platelet ADP-induced activation is regulated by the P2Y12 and P2Y1 signaling pathways. We have previously shown that blockade of the P2Y12 signaling pathway in a murine model of sepsis improves outcomes in male mice. On the other hand, P2Y1 deficiency had no effect on inflammation levels in a murine model of sepsis. We have shown that by blocking specific signaling pathways in platelets we can regulate inflammation without compromising platelet functions in male mice. This project aims to investigate further the mechanism through which purinergic signaling in platelets contributes to sepsis in both sexes. The project is investigating sepsis which is a complex clinical syndrome with high mortality and currently, there are no treatments.
|14."Use of an engineering testbed of bone metastasis of cancer to evaluate therapies" - Kalpana Katti, University Distinguished Professor, Civil, Construction & Environmental Engineering|
Both prostate cancer and breast cancer have the propensity to migrate to bone at which point the cancer is incurable. Anticancer drugs are ineffective and often only palliative treatment is possible. Animal models fail since the animal dies before metastasis to bone occurs and human samples are difficult to obtain since patients are in a hospice care when bone metastasis has occurred. Skeletal or bone fractures are common in patients with breast and prostate cancer metastasis. We have designed a novel test bed of bone site that replicates the morphology, chemistry and biomechanics of the bone site. This bone-mimetic is used to create bone metastasis tumors of breast and prostate cancer using commercial and patient derived human cancer cells. We use this testbed to screen drugs, design therapies and evaluate fundamental characteristics of the cancer. This project also has an extensive computational component that involves modeling at molecular, and cellular level. The flow of blood is simulated through use of a specially designed bioreactors that enables shear stresses on the growing tissue that simulates blood flow.
|15. "Cooperative Catalysis to Improve Efficiency of Organic Reactions: Synthesis of Warfarin Analogs" - Mukund Sibi, University Distinguished Professor, Chemistry and Biochemistry|
The installation of all carbon quaternary centers with control over relative and absolute stereochemistry persists as a formidable synthetic challenge in organic chemistry. One goal for our laboratory is to develop an achiral-template-driven strategy to deliver reliable and effective solutions for the installation of hindered chiral centers by utilizing several important synthetic transformations. Preliminary results from our laboratory clearly demonstrate the identification of a novel achiral template containing well-defined sites for interactions with different chiral activators (Lewis acids, Bronsted acids, etc.). Work for the 10 week summer program involves the synthesis of Warfarin analogs. Warfarin is a common therapeutic used to treat blood clots. Although warfarin is prescribed as a racemate for thrombosis, the pharmacological properties of the enantiomers are different. Enantioselective synthesis of warfarin analogs is of interest to the synthetic community. Summer Work: The conjugate addition of nucleophiles to b,b-disubstituted-a,b-unsaturated systems (1) and a,a,b-trisubstituted-a,b-unsaturated acceptor olefins is non-existent in the literature. This study will underscore the formation of all-carbon quaternary centers (enantioselective synthesis–quaternary centers containing trifluoromethyl group) as part of an approach to access warfarin analogs with two contiguous stereocenters with diverse functionalization. The summer student will investigate and optimize methods for the efficient synthesis of fluorine containing warfarin analogs (see scheme below). The summer student will learn hands on techniques to perform organic reactions, isolation, purification, and characterization of the products. The student will become acquainted with different spectroscopic techniques used in structural characterization of organic compounds. The REU student will work closely with a graduate student and learn to work in a team setting. The project is well suited for training the summer student in a variety of soft skills.
|16. "Modeling the cell-cell adhesion in breast cancer tumors" - Dinesh Katti, Professor, Civil, Construction & Environmental Engineering|
Breast cancer is one of the leading causes of death due to cancer in women. In my group, we have used a synergistic approach to study cancer progression leading to tumor formation using complimentary experimental and modeling tools. In the current work we will be using data from our modeling work on adhesion proteins, experimental work on cell-cell adhesion and 3D invitro models of cancer progression to build cancer cell models to simulate cell-cell adhesion leading to tumor formation. The computational modeling effort will lead to better understanding of the mechanisms responsible for formation of tumors. Some of the modeling results will be verified by experiments using a unique atomic force microscopy facility in the mentor’s laboratory. The undergraduate student will be trained and guided by graduate students working in the field and by the faculty mentor. The student will be using advanced computational tools for the study, but does not need to have prior experience. The specific outcomes of the research include modeling cell-cell binding, evaluating binding strength, and evaluating changes to cell morphology due to cell-cell adhesion as cancer progresses.
|17. "Psychosocial factors, sleep, and cardiometabolic health" - Katherine Duggan, Assistant Professor, Psychology|
The focus of this research assistantship is to gain foundational training in behavioral sleep medicine, including the data collection, scoring, and analysis of multidimensional sleep health data. This includes self-reports (for example, of sleep quality) and behavioral measures from actigraphy (actigraphs are small, wrist-watch like devices which track patterns of movement and light exposure and from that information identify the beginning and end of sleep periods). Trainees will be able to go in-depth and score and analyze sleep data in one of the lab’s ongoing studies, including: (1) a clinical trial for the effects of goal setting on sleep and cardiometabolic health; (2) an observational study investigating the prevalence of clinically-significant sleep problems and hypertension in low-income factory workers; or (3) a longitudinal study of sleep and resilience in American adults during the pandemic. Expected outcomes for students from the research experience: The trainee will receive foundational training in behavioral sleep medicine, cardiovascular behavioral medicine, and health psychology. They will also learn to administer, score, and interpret sleep questionnaires and data. Additionally, the trainee will identify a topic of interest related to sleep health (e.g., psychosocial factors, cardiovascular health, sociodemographic variables), and will develop a pre-registered data analysis plan for their topic in one of the previously described studies. The trainee will be mentored in statistical analysis and will submit an abstract based on this work. Finally, the trainee will have the opportunity to turn their work into a manuscript submission for a peer-reviewed empirical journal, with the goal of submission towards the end or after the Summer Undergraduate Biomedical Research Program.
|18. "Innovative Pathogen Disinfection Technology by Coupling Far UVC and Peracetic Acid" - Jiale Xu, Assistant Professor, Civil, Construction & Environmental Engineering; Syeed Iskander, Assistant Professor, Civil, Construction & Environmental Engineering|
Opportunistic infection caused by emerging pathogens including bacteria, viruses, and protozoa to people with certain medical conditions occurs more frequently in recent years, especially during the pandemic period of SARS-CoV-2 (COVID-19). When pathogens that come from hospital wastewater (pool of pathogens) and municipal wastewater (containing fecal pathogens) are not completely inactivated, they are potentially present in source water and even in drinking water systems, posing a high health risk to humans. Common pathogen disinfection technologies employ chemicals, such as chlorine, chloramine, chlorine dioxide, and ozone, or high-energy ultraviolet light (UV). However, these disinfection schemes feature several major drawbacks. First, they are not effective for several pathogens, such as cryptosporidium, one of the most problematic water-borne pathogens. Second, chlorine-based chemicals and ozone can unintentionally generate toxic disinfection byproducts, a group of cytotoxic, genotoxic, and carcinogenic chemicals. Lastly, disinfection loses its effectiveness at low temperatures, and the chemical or energy cost is high for cold regions, such as the Fargo area. Hence, an effective, efficient, and low-cost pathogen disinfection technology is warranted, especially for emerging pathogens, such as viruses. This project proposed to couple two innovative disinfectants, far UVC and peracetic acid, for advanced pathogen disinfection with synergistic benefits. Far UVC is the UV light at 222 nm that can damage the nucleic acids of pathogens, which exhibits 2–12 times higher inactivation for pathogenic viruses than low-pressure mercury UV (LPUV) lamps, the most prevalent UV setup for pathogen disinfection. Particularly, it provided the highest disinfection for SARS-CoV-2. Additionally, far UVC features the many other advantages of being mercury-free, safe for mammal eyes and skins, and thermally stable at 0–5 °C. However, far UVC suffers from medium-level disinfection for bacteria. Peracetic acid as a new disinfectant provides excellent performance in killing common bacteria by oxidizing proteins in pathogens, and it can also prevent the formation of toxic disinfection byproducts. The benefits of combing these two technologies include effective damage to a wide range of pathogens, no byproducts, low cost, and high adaptability to cold regions. The undergraduate will work on developing this combined technology for pathogen disinfection for model pathogens in hospital wastewater, municipal wastewater, and drinking water. The undergraduate research will be expected to obtain: 1) the efficiency of coupling far UVC and peracetic acid in inactivating model pathogens in the comparisons with other technologies, 2) the optimal conditions of this technology in disinfecting pathogens, and 3) the performance in treating real wastewater samples.