Cohort Four Projects

Departments of Faculty mentors

Chart

Majors of undergraduate participants

29%  Biology
47%  Interdisciplinary majors
Biochemistry, Neuroscience
Bioengineering
26%  Engineering
Computer Science, Physical Sciences
  • 15 Interdisciplinary Research teams comprised of faculty, graduate students and undergraduates, with half of each team from life sciences and half from other STEM disciplines
  • Ten of the 27 faculty mentors were assistant professors
  • Of 34 undergraduates, 35% were juniors, and 65% were seniors
  • 35% of the students were under-represented in their disciplines
  • 18% were from underrepresented minority groups
  • 18% were women with majors in engineering, geology, physics and chemistry
  • 27 graduate students participated as research mentors to the undergraduates
  • Funds from SUNY Research Foundation supported two students

2014-15 Project Descriptions

Project Titles

1. Role of Biofilm Formation in the Functionality of Sensors | Read more

2. Extension of Statistical-Computer Based Technology in Developing a Clinical Decision Support System for Malignant Pleural Effusion Analysis | Read more

3. Investigating the Pharmacokinetics of pHLIP Conjugates and the Effect of Flow Shear on pHLIP – cell Interactions Using in vitro Models | Read more

4. Ancient Microorganisms in Fluid Inclusions in Halite and Gypsum | Read more

5. Lyme Disease EpidemiologyRead more

6. Image Processing to Characterize In Detail the Spatial Properties of Multiple Bacterial Species
within Biofilm
| Read more

7. Use of Engineered Nanovesicles to Investigate Formation and Biological Function of Bacterial Outer Membrane Vesicles | Read more

8. Investigating the Influence of Features, Intensity and Viewpoint on Classification of Expressions in Children or Adult | Read more

9. Sequence Selective Recognition and Imaging of Double Stranded RNA via Triplex Forming PNA | Read more

10. Determination of Glutamate Transporter Involvement in Ethanol Withdrawal related Neuronal Excitotoxicity | Read more

11. Development of a Nanodelivery System for Enhanced Treatment of Biofilm-related Infections | Read More

12. The Effect of Bacteria on the Structural Failure of Bronchial Epithelial Cells | Read more

13. Food Additives, the Gut Microbiome and Metabolic Disorders | Read more

14. Investigating the Role of Shear Stress in Biofilm Development Using Microfluidic Technology | Read more 

15. Human Traffic Impacts on Microbiological Metal Cycling in Roadside Soils and on Biodiversity in Urban Streams | Read more 


Project Descriptions:

1. Role of Biofilm Formation in the Functionality of Sensors

Faculty Mentors: Dr. Karin Sauer, Professor, Biological Sciences and Dr. Wayne Jones, Professor, Chemistry

Graduate Mentors: Amy Chen, Chemistry, Bandita Poudyal, Biology

The HHMI research project brings together a chemist Wayne Jones and a microbiologist, Karin Sauer, to explore capabilities of the bacterium Pseudomonas aeruginosa to form surface associated communities called biofilms. The Jones group has designed new conjugated fluorescence turn-on polymers which capitalize on recent discoveries in inorganic/organic hybrid systems. The systematic characterization of the selectivity and sensitivity for these materials with known toxic metal ions in aqueous solution (Fe2+/3+, Hg+, Crn+, Pb+/2+, and Cd+/2+) will lay the ground work for next generation sensors for application to anions, toxic small molecules, or biomarkers of interest to the environmental health science community. Preliminary studies have demonstrated great success with one such "turn-on" organic/inorganic hybrid polymer chemosensor; tmeda-PPETE/Cu2+ was found to be highly sensitive (10 ppb) and selective for Fe2+ and Fe3+cations in hydrophilic solution. However, the functionality of these sensors in environmental and industrial settings depends on the surface of sensors remaining intact and not being subject to biofouling, a process involving the accumulation of bacteria (biofilm formation) on sensor surfaces.

Our primary objective is to develop the next generation of fluorescent polymeric organic/inorganic hybrid chemosensor materials which enable us to modulate the extent of biofouling. This will be achieved by determining which sensor properties (type of polymers and material composition, fiber thickness) enhance or impair bacterial attachment and subsequent biofilm formation. Attachment and biofilm formation will be analyzed by viable count, microscopy including fluorescent and confocal microscopy, and various biochemical assays. Additionally, the functionality of the sensors will be used as an independent measure of biofouling. Sensors including turn-on fluorescent polymer chemosensors (FCP's) with improved selectivity for Fe2+/3+ will be analyzed by UV-vis, FTIR, NMR, and Electron Microscopy to explore functionality and the oxidation state of Fe in these systems.

Academic preparation for undergraduate students:

One student with a chemistry background and interest in analytical chemistry will participate in the research project by synthesizing new conjugated fluorescence turn-on polymers which incorporate an inorganic/organic hybrid system as well as other techniques for identifying Fe2+/3+. The student will use organic synthetic techniques to prepare the polymers. The student will also investigate the photophysical characteristics of the polymers using UV/VIS and emission spectroscopies. Ideally, this student will have completed the course in Organic Chemistry.

One student with a biology background and interest in microbial biofilms and/or biochemistry will participate in the research project. The student will use microbiological approaches to grow bacteria including aseptic techniques and viability determination assays, as well as various other microbiological and biochemical techniques. Ideally, this student will have completed the course in Microbiology.

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2. Extension of Statistical-Computer Based Technology in Developing a Clinical Decision Support System for Malignant Pleural Effusion Analysis

Faculty mentors: Dr.Walker H. Land, Jr. Bioengineering, Dr. David Schaffer Bioengineering and Dr. Xingye Qiao, Mathematics

Graduate Student Mentor: Yinglei Li.

The HHMI research project brings together Research Professor Walker Land , Dave Schaffer , Xingye Qiao, Dr. Sidhu (UHS), Yinglei Li and Martha Nelson (UHS) who have been and are developing statistical learning theory (SLT) paradigms that result in complex adaptive systems (CAS), which measure the efficacy of drug treatments, survivability analysis of patients with operable metastatic lung cancer, and developing methods to intelligently process covariant features using kernel density estimates as applied to cancer screening and diagnosis. Specifically this project involves developing a Clinical Decision Support System (CDSS) for Malignant Pleural Effusion Analysis

A prototype CAS system based on SLT was designed, implemented and tested to validate the theories on which the design was based. The test cases were "cherry picked" isolated epithelial   cell nuclei that were successfully and accurately classified in the manner described below, thereby verifying the overall SLT design of a composite serial combination of intelligent system components. The premise is  that if the intelligent CAS could not successfully classify individual perturbed nuclei, it would not classify clustered nuclei.

Specifically, the validation component of this preliminary design produced the following conclusions (the reader is reminded that these conclusions apply to isolated epithelial cell nuclei and not the more complex clustered and overlapping nuclei): The purpose of this initial research effort is to develop a prototype CDSS to aid pathologists in correctly discriminating reactive benign mesothelial from malignant epithelial cells. Images of stained pleural effusion slides were collected by Dr. Jagmohan Sidhu. The project has provided: (1) generation of a quantified dataset derived from the nuclei of cells found in pleural effusion images; (2) an intelligent system capable of classifying visually known cases which was validated using ROC analysis (AZ around 0.90); (3) an intelligent system with the potential to classify atypical cells; (4) an assembled CDSS capable of accepting images from new cases and rendering output to a clinician. In doing so, this project has laid a framework for the quantitative use of pleural effusion images in the development of second opinion clinical decision support systems.

Internal recommendations have been provided to guide future work with the intention of further validating the clinical usefulness of the system, its theoretical underpinnings, as well as to increase the quality and proper use of current and future data. Clearly, more data accompanied by patient outcomes and other experimental information will be critical to validating and developing the CDSS.

We are happy to report that this research effort resulted in the following accepted paper:

"Clinical Decision Support System (CDSS) for the Classification of Atypical Cells in Pleural Effusions" David Bassena, Saurabh Nayaka, Xia Chong Li,a Mitchell Sam,a Jagmohan Sidhu, MDb, Martha F. Nelson, RN MS MPA,b Walker H. Land, Jr.aa Dept. of Bioengineering, Binghamton University Vestal, NY 13902 USA b UHS Wilson Medical Center, Johnson City, NY 13790 USA, to be presented at the CAS symposium ,Nov. 13-15 th, as well as being published on –line by Procedia Computer Science, at a to be specified URL.

The current activity involves : One team is currently working on creating a dataset of the visually benign and malignant images. This is done through image processing, which must do two things: (1.) automatically identify nuclei and (2.) separate touching/occluded nuclei. They have created a preliminary dataset of "automatically" found nuclei of the benign and malignant images, and are working on now declustering the remaining groups. Next semester their plan is to process the atypical cell images, adjusting their image processing techniques as necessary.

The objective of this follow on effort is to integrate the current activity (cover the more difficult images) into a clinical prototype to be used as a second opinion clinical decision support system (CDSS) in a clinical environment as specified by Dr. Sidhu and his colleagues.

System Process:

The process will continue to be data collected by Dr. Sidhu from his data base of plural effusion patients and presented in a image format for image processing by the Intelligent CAS.

Academic Preparation:

Ideally students should have a background in mathematics, bioinformatics, image processing, statistics and programming. A background in Machine Learning would be also helpful. Generally, students who have completed BME340 and BME302 who have demonstrated a capability for performing research using real biomedical data have been the successful performers on this project. The data we get are de-identified at UHS, so students never encounter identifiable patient information.

Mentoring arrangements:

Prof. Land (with assistance from Profs. Schaffer and Qiao) along with Yinglei Li will monitor several undergraduate students the design, programming, testing and evaluation of all experiments developed to test / evaluate the efficacy of new / modified SLT paradigms that result in new CAS. Dr. Sidhu and Martha Nelson will monitor all clinical results / conclusions / interpretations resulting for these research experiments.

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3. Investigating the Pharmacokinetics of pHLIP Conjugates and the Effect of Flow Shear on pHLIP – cell Interactions Using in vitro Models

Faculty Mentors:

Dr. Gretchen Mahler, Assistant Professor, Bioengineering

Dr. Ming An, Assistant Professor, Chemistry

Graduate Student Mentors

Courtney Sakolish, Bioengineering

TBA, Chemistry

This HHMI research project brings together a bioengineer, Gretchen Mahler, and a chemist, Ming An, to investigate the pharmacokinetics of pHLIP conjugates.

Cancer chemotherapy is limited by toxic effects in healthy tissues, motivating the search for targeted drug delivery. We have developed an approach to target acidic solid tumors using the pH-(Low) Insertion Peptide (pHLIP) (Figure 1a)1. We have shown that pHLIP can translocate phalloidin, a membrane-impermeable polar toxin (Log P-2), into cancer cells to inhibit proliferation in vitro (Figure 1b)2. In collaboration with the Mahler lab, work proposed here aim to investigate the binding and pharmacokinetics of pHLIP conjugates using physiologically realistic microfluidic models.

While pHLIP is a promising system, studies in mice revealed the following shortcomings that point to directions of further studies:

state 2 image(a) Phalloidin is probably not a sufficiently potent cytotoxic agent in vivo.For this reason, we will synthesize pHLIP-conjugates using cytotoxic agents far more potent than phalloidin, such as auristatin, maytansinoid DM4, and CC-1065/adozelesin analog DC1. Unlike phalloidin (Log P ~ -2), these toxins are lipophilic (Log P ~ 1-5) and membrane-permeable. Attaching pHLIP to these toxins serve two purposes in vivo: (1) pHLIP translocation may circumvent capture of the drug by P-glycoproteins —the drug efflux pumps responsible for multi-drug resistance in cancer cells; and (2) at neutral pH, pHLIP may partition to the cell surface, but will not insert, effectively blocking toxin entry into healthy cells (and reducing off-site toxicity). The microscale cell culture analog (µCCA) model seems the perfect setting for studying the following question: How does flow shear influence pHLIP-cell interactions?

(b) Pharmacokinetics of pHLIP monomer is poor. More than 90% of pHLIP peptides are lost within 24 hours. The tissue models will allow us to probe the metabolic fate of pHLIP and pHLIP-conjugates in more detail (by answering questions such as 'Are pHLIP peptides cleaved during circulation after i.v. injection?' or 'Are pHLIP peptides specifically metabolized by certain cell populations?').

(c) The pHLIP peptide targets acidity in vivo. Thus, acidic tissues other than tumors will also be targeted, such as the kidneys (as we observed), which may lead to toxic side effects in pHLIP-mediated drug delivery. We propose to synthesize pHLIP-PEG conjugates in the size range of ~ 5-10 nm in hydrodynamic diameter (M.W. 30-100 kD). If the µCCA system can accurately model renal filtration (the size of the renal pore is ~ 5 nm), then the pharmacokinetics of pHLIP-PEG conjugates may be investigated.

The early stages of drug development often involve animal testing. This process is expensive, lengthy and at times, controversial. In addition to these issues, there remains the question as to whether the data obtained from these animal tests can be extrapolated to human systems. To save time and money, µCCAs can be utilized to accurately model human systems and allow for more directed pharmaceutical testing. The target cell line used in this project is HK-2, a line of immortalized human kidney proximal tubule cells. The proximal tubule is the region of the kidney responsible for the reuptake of salts, water and simple sugars from the urine to be reused elsewhere in the body; and also a site of pHLIP accumulation. Additionally, the effects of shear stress on these regions have not been fully characterized, making this an interesting target for the creation of a microfluidic device with an incorporated pH gradient and fluid shear stress to closely mimic in vivo conditions. The results of our preliminary work suggest that with further characterization and testing, HK-2 cells may be able to accurately model the human renal proximal tubule in vitro. Work over the summer will include seeding of HK-2 cells into a multi-channel microfluidic device with incorporated pH gradient and fluid shear stress to further characterize this cell line. One of the goals of this project is to develop kidney microfluidic device that can describe the binding events, mechanism of action and toxicity of circulating pHLIP and pHLIP-toxin conjugates.

Academic preparation for undergraduate students

One student in bioengineering will participate in the project by developing and characterizing a kidney microfluidic device for studying pHLIP. Ideally, the student will have BIOL 117-118 and BE 203 completed. One student in chemistry will help to synthesize pHLIP-conjugates. This student should have completed a course in organic chemistry.

References

1. Andreev, O. A. et al. Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo. Proceedings of the National Academy of Sciences 104, 7893-7898 (2007).

2. An, M., Wijesinghe, D., Andreev, O. A., Reshetnyak, Y. K. & Engelman, D. M. pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation. Proceedings of the National Academy of Sciences 107, 20246-20250 (2010).

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4. Ancient Microorganism Communities in Fluid Inclusions in Halite and Gypsum

Faculty Mentors:

Dr. Tim Lowenstein, Geology and Dr. Koji Lum, Anthropology/Biology

Graduate Student Mentors:

Emma McNulty , Geology and Yue (Amy)Zhang, Biology

The HHMI research project brings together anthropologist/biologist Koji Lum and geoscientist Tim Lowenstein. We are funded by NSF for the project "Ancient Microorganism Communities in Fluid Inclusions in Halite and Gypsum"; the first paragraph of the summary is copied below:

The proposed research explores the fundamental problem of long-term survival of microorganism communities and preservation of biomaterials in fluid inclusions in halite and gypsum. The goal of the proposed research is to obtain data on the distribution, survival, and diversity of microorganism communities and biomaterials that have been in the subsurface for periods of thousands to hundreds of millions of years. It will involve systematic examination of the state of survival and preservation of the suite of microorganisms, including prokaryotes (Bacteria and Archaea), eukaryotes (algae, fungi) as well as DNA and other biomolecules (i.e., chlorophyll, carotenoids, glycerol) in fluid inclusion ecosystems in halite and gypsum. The research plan will follow the successful interdisciplinary approach recently used for the study of halite from the subsurface of Death Valley, CA, but extended to older halite deposits, 105 to 108 Ma in age, and for the first time to gypsum. New emphasis will be placed on: (1) Molecular biological techniques involving amplification of fragments of DNA by the polymerase chain reaction (PCR), followed by cloning and sequencing, which will characterize the phylogenetic diversity of microorganisms in fluid inclusions in saline minerals. (2) Raman spectroscopy, which has the potential to quantitatively characterize the nature of biomolecules in fluid inclusions, in situ.

Undergraduate project will involve (1) Microscopy (transmitted and UV light, Scanning Electron Microscopy) documenting the microbial population and biomaterials trapped within modern and ancient crystals of gypsum, and the sodium carbonate minerals trona and nahcolite, and (2) Molecular biological techniques involving amplification of fragments of DNA by the polymerase chain reaction (PCR), followed by cloning and sequencing, which will characterize the phylogenetic diversity of microorganisms in gypsum, trona and nahcolite.

Projects and academic preparation for undergraduate students:

- A biology/biochemistry/biological anthropology undergraduate student will carry out the molecular biological techniques to characterize the phylogenetic diversity of the microorganisms as described above. This will require completion of Introductory Biology, Microbiology (preferably with laboratory) and a course in genetics (e.g. Anth 428).

- A geoscience sophomore or junior who has completed an Introductory class in Geology, Mineralogy (GEOL 212), Earth History (GEOL 213), and Earth Surface Processes (GEOL 211). Upper level courses such as Paleobiology (GEOL 366) and Geochemistry (GEOL 470), and Geomicrobiology (GEOL 460) are recommended but not required. Introductory Biology will be an asset, but not required. This student will be focusing on microscopic techniques such as transmitted light microscopy and scanning electron microscopy toward recognition of microorganisms and biomaterials in fluid inclusions.

Mentoring arrangements:

Yue Zhang, Ph.D. student in the laboratory of J. Koji Lum will mentor the student doing the molecular biological work. Ph.D. student Emma McNulty will mentor the geoscience student.

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5. Lyme Disease Epidemiology

Faculty Mentors:

Dr. Ralph Garruto, Professor, Anthropology

Dr. Hiroki Sayama, Associate Professor, Biomedical Engineering

Dr. Julian Shepherd, Associate Professor, Biological Sciences

Rita Spathis, Laboratory Director, Biomedical Anthropology

Graduate Student Mentors:

John Darcy, Anthropology Jeff Schmidt, Bioengineering

This HHMI research project brings together biomedical anthropologist Ralph Garruto, complex systems scientist Hiroki Sayama, and ecologist Julian Shepherd using ecological, spatial, and human behavioral and demographic data to construct a computational model to predict risk of tick-borne disease infection in humans living, working, and recreating in and around built environments (suburban university, parks, and suburban and peri-urban spaces

Emerging and re-emerging infectious diseases in developed and developing nations where chronic disease is on the rise is a hallmark of the 4th epidemiologic era. Diseases such as Dengue Fever, West Nile Virus and Malaria are re-emerging in areas where either they had previously been eradicated or did not exist at all. Over the last four decades new diseases such as HIV-AIDS, Ebola hemorrhagic fever and Lyme disease are the result of new human-environment interactions. Lyme disease emerged in the mid 1970's and has become the most common vector-borne disease in the United States today, with an estimated 300,000 cases annually (CDC, 2013). The BU Lyme and tick-borne disease research group is investigating the emergence of this disease in upstate New York and other areas of the Northeastern U.S. The disease occurs in humans when an infected tick comes into contact with humans or other animals and attaches itself for a blood meal.In the process of feeding, the tick transfers a gram negative bacteria, Borrelia burgdorferi, the infectious pathogen.Lyme disease ecology and evolution is driven by geographic, ecologic, climatic, and human land use patterns as they relate to the Lyme disease vector, Ixodes scapularis (black legged tick or deer tick). Other human pathogens, anaplasma and babesia are also carried by I. scapularis and are also being studied by the team. In the coming year the Lyme disease project will also seek to expand its analytical framework to other tick-borne diseases such as anaplasmosis and babesiosis to investigate the potential role co-infections play in Lyme disease epidemiology within built environments.

We are currently using the Binghamton University campus as a natural experimental model to study the factors involved in Lyme disease transmission in a small geographically defined area. The project centers on five strategic areas (field ecology, laboratory analysis, field behavioral study, clinical symptomology and mathematical modeling) that are designed to address the factors influencing Lyme disease and other tick-borne infection emergence in human populations.  The field component of the project involves the collection of ticks from various microecologies on campus using a corduroy cloth to drag the leaf litter and lower vegetation. Thus far we have collected and tested over 1000 ticks from the university campus and the local region. In the laboratory component of the project, total DNA is extracted and the presence of B.burgdorferi, A. phagocytophilum, and B. microti is determined by PCR amplification and cycle sequencing.  Demography, human traffic patterns, the built environment and human behaviors are also currently being assessed to determine exposure and risk that will lead to subsequent public health strategies and interventions.  The reservoir host for the tick-borne agents is the white footed mouse, Peromyscus leucopus, is being trapped to determine the prevalence of infection in this tick-borne competent reservoir.

The overall modeling of tick-borne disease risk factors is the end stage research component of this project. Modeling of ecological dynamics of Lyme disease is an emerging subfield within the broader context of Lyme disease epidemiology. Models relating to vector-host-environment interactions are at the core of understanding the interrelating variables involved in disease transmission to humans. Clinical diagnosis of Lyme disease is heavily reliant on serologic and objective testing and doesn't necessarily incorporate ecological factors. Current Lyme disease laboratory diagnostic measures can be subject to error as well as untimely with a window of effectiveness which peaks four weeks after infection. We propose utilizing variables derived from existing models combined with epidemiological, ecological, social and behavioral factors to formulate a standardized algorithm of risk for use with public health settings.

Academic Preparation and Student Participation

The project is multifaceted and the student team and faculty interdisciplinary. Students will be trained in the proper conduct of research, in the search of the relevant scientific literature, in building of databases, in data preparation and analysis, in mathematical modeling and computer programming for numerical simulation and statistical analysis, and in communication of the results (both written and oral) to all team members and a final product to the scientific community and public health departments locally and at the state level, and perhaps nationally.

Graduate Student Mentoring Arrangements

BiologicalAnthropology graduate student John Darcy (supervised by Ralph Garruto) will mentor the undergraduate students in behavioral, ecological, demographic, epidemiological and clinical risk factors associated with the modeling.

Biomedical Engineering graduate student Jeff Schmidt (supervised by Hiroki Sayama) will mentor the undergraduate students in the mathematical/computational modeling of the risk factors of Lyme disease and the epidemic dynamics.

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6. Image Processing to Characterize In Detail the Spatial Properties of Multiple Bacterial Species within Biofilm

Faculty Mentors:

Dr. Claudia N.H. Marques, Biology and Dr. Scott Craver, Electrical Engineering

Graduate Student Mentors:

Elin Mina, Biology and Alireza Farrokh-Baroughi, Electrical Engineering

The BU-HHMI research project brings together microbiologist Cláudia N. H. Marques, electrical engineer Scott A. Craver to analyze the biofilm development of bacterial infections on Eukaryotic epithelial cell lines.

The proposed research will investigate the dynamics of the multi species biofilm populations within Eukaryotic epithelial cell lines. Microbial infections, including nosocomial infections are mainly caused by multi-species bacterial populations. However, the available treatments are based on studies of single species bacterial populations. Currently not much is known about the initial stages of infection and how several bacterial species interact and eventually develop into biofilm within a Eukaryotic cell. Understanding the development of mixed species biofilms within epithelial cells could lead to a significant improvement on the efficacy of the treatment of infections.

Currently the electrical engineering side of the project has developed a simulation framework of biofilm development, based on bacterial cell growth. This year we will start implementing, on the model, other conditions that contribute to biofilm development such as: cell locomotion, adhesion of the bacteria to the surface and to one another, and the effects on their composition upon exposure to antimicrobials. This research will involve the tracking of individual bacterial cells, to better understand the mechanisms by which they attach to a Eukaryotic cell and the interaction of the two bacterial species within the biofilm population and with the epithelial cells. Biofilms composed of Pseudomonas aeruginosa and Staphylococcus aureus will be used to co-infect Eukaryoticepithelial cells. We will monitor the development of biofilms throughout the infection of eukaryotic cells when using continuous culture system. This monitoring will be performed using microscopy and cell viability. Image processing, estimation and optimal hypothesis testing algorithms will be employed to accurately estimate population density and distribution of different bacterial species in the spatial domain and to develop a predictive model for the effect of local population density of one species versus another. This includes the continued development of a software product for analyzing bulk image sets acquired using confocal microscopy. Once the basic methods are established further research will be done to establish the efficacy of certain antimicrobials in biofilm killing and the change in shifts in population dynamics occurring throughout the treatments.

A biology student will carry out the culturing of the microorganisms and the molecular and microscopic techniques necessary to develop the project. This will require the completion of Biol 118 and preferably the completion of Microbiology (Biol 224 or 314).An Electrical and Computer Engineering student with expertise in signal and image processing will carry out the analysis and write computer software. This will require the completion of Signals and Systems (EECE 301) and skill in computer programming. Introductory Biology would be an asset but not required.

Mentoring Arrangements: Dr. Cláudia N. H. Marques and graduate student Elin Mina will mentor the student working on bacterial biofilms. Graduate student Alireza Farrokh-Baroughi and Dr. Scott Craver will mentor the Electrical and Computer Engineering student.

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7. Use of Engineered Nanovesicles to Investigate Formation and Biological Function of Bacterial Outer Membrane Vesicles

Faculty Mentors:

Dr. Jeffrery Schertzer, Biology andDr. Paul Chiarot, Mechanical Engineering

Graduate Student Mentors:

Adam Cooke, Biology and Li Lu, Mechanical Engineering

This HHMI research project brings together a biologist, Jeffrey Schertzer, and an engineer, Paul Chiarot, to investigate the mechanism of bacterial Outer Membrane Vesicle (OMV) formation and the role of these vesicles in biofilm formation.

image of OMVs, how they are formed and the nature of their functionsVesicle-mediated transport was once believed to be an exclusive feature of eukaryotic systems. It is now understood that Gram-negative bacteria also have the ability to sort, package and transport cargo to other cells in vesicles that bud off from their outer membrane (OM). This mode of transport can provide target specificity, allow for concentrated long-distance delivery and provide protection from the environment, including elements of the immune system. As a testament to their versatility and importance, OMVs have been shown to play important roles in the delivery of virulence factors, modulation of the host immune system, bacterial cell-to-cell communication (termed quorum sensing, QS), and the maturation of biofilms. For this reason, it is of great interest to understand how OMVs are formed and the nature of their functions in bacterial physiology and behavior.

In the opportunistic human pathogen Pseudomonas aeruginosa it was shown that the OMV-transported QS signal PQS (Pseudomonas Quinolone Signal) is responsible for inducing the formation of the very OMVs into which it is packaged [4], even in the absence of its receptor or de novo protein synthesis. This suggested that the OMV-inducing characteristic was based on a physical effect of PQS and not upon its role in altering gene expression as a QS signal. Following this, Dr. Schertzer proposed a mechanism (Fig 1) by which insertion of PQS into the outer leaflet of the outer membrane would expand that leaflet relative to the inner one and thereby induce membrane curvature, eventually leading to bleb formation and finally release of OMVs [5]. This work successfully showed that PQS could induce curvature in model phospholipid membranes, but suffered from the caveat that it could not be tested using artificial membranes matching the asymmetric lipopolysaccharide (LPS) / phospholipid (PL) (outer leaflet / inner leaflet) architecture of the bacterial OM. This was an unfortunate limitation of technology since no such membrane structures have been made at the appropriate size or material scale to test these questions. Dr. Chiarot's nanoscale vesicle technology (below) promises to alleviate this problem, allowing OMVs of controlled size to be fabricated with lipid content and architecture identical to that of the natural OM. The proposed work will use large (bacteria-sized) engineered OMVs to test the applicability of the bilayer-couple model to bacterial surfaces. Nanoscale (50 – 500 nm) OMVs will be used to investigate the preference of PQS to insert into membranes of different curvature (as in [3]). That PQS might prefer curved membranes is a hypothesis developed in our group to explain the apparent ability of PQS to self-aggregate in the OM to be packaged into OMVs – greater than 80% of PQS in a bacterial culture is associated with OMVs. In addition, by engineering the OMVs to have the opposite asymmetry (LPS = inner, PL = outer), we will use engineered OMVs to screen for compounds that can expand the PL leaflet, which would antagonize natural OMV production and hinder bacterial virulence.

The proposed work also aims to define the role of OMVs in biofilm maturation. Biofilms are communities of bacteria that have attached to a surface and encased themselves in a structural and protective extracellular matrix. In this mode of growth, bacteria more resemble a multicellular tissue than the solitary planktonic cells that they are commonly thought to be. In fact, it is now appreciated that biofilm growth is the common mode of growth for bacteria in nature and biofilm infections make up the majority of bacterial infections. When PQS production is disrupted in P. aeruginosa, the organism fails to form mature biofilms [1], but it is unclear whether this is due to the loss of the QS function of PQS or the potential structural function of OMVs in the biofilm. Since PQS physically stimulates the formation of OMVs in P. aeruginosa, it is exceedingly difficult to generate natural OMVs that lack PQS to attempt to discriminate between these two functions. In addition to investigating PQS-free OMVs from other species, we will use engineered OMVs to attempt to complement biofilm maturation deficiencies in PQS biosynthetic mutant strains. These OMVs will contain no PQS or bacterial protein and will serve as the perfect 'blank slate' to add back individual components and identify key contributors to biofilm maturation.

Fig 2 – Proposed microfluidic device. Emulsions flow from left to right in the image. Lipid monolayers are formed on the emulsion surface and at the interface of co-flowing streams. A bilayer is formed when the emulsions are pushed through the interface of the co-flowing streams using the dielectrophoretic (DEP) force.

op1 -2

Fig 2 – Proposed microfluidic device. Emulsions flow from left to right in the image. Lipid monolayers are formed on the emulsion surface and at the interface of co-flowing streams. A bilayer is formed when the emulsions are pushed through the interface of the co-flowing streams using the dielectrophoretic (DEP) force.

The use of microfluidic technology is an attractive option for producing customized synthetic vesicles. This technology has many important advantages, including: precise control over dispensed volumes, high-throughputs, and repeatability. The internal volume of a microfluidic device is on the order of nanoliters, while typical solution flow rates can be as low as picoliters-per-minute. Smaller volumes mean less material (i.e. lipid) is consumed – a significant issue for high-cost lipids such as LPS and a true hurdle overcome using this technique. Most notably, our proposed technique will be capable of building vesicles with asymmetric membranes, where each leaflet is composed of a different lipid. This is an essential requirement for each of the downstream studies.

Our proposed technique uses liquid emulsions and lipid self-assembly inside a microchannel network built using a layer of poly-dimethylsiloxane (PDMS) bonded to a glass substrate with patterned electrodes [2]. This system is ideal for achieving control over vesicle unilamellarity, size, and uniformity. Membrane curvature (proportional to vesicle size) will be tightly controlled, with both narrow and wide OMV size distributions possible. This feature is an excellent fit for downstream applications as both wide distributions (for studying PQS insertion into OMVs of different curvature) and narrow distributions (for biofilm complementation experiments) will be called for. Our strategy for forming vesicles relies on four key steps as shown in Fig 2: (i) emulsion formation using flow focusing, (ii) formation of co-flowing laminar streams (iii) lipid monolayer self-assembly at multiple liquid-liquid interfaces, and (iv) dielectrophoretic steering to transfer lipids to the emulsion surface. Lipid bilayer self-assembly takes place at the surface of the emulsions, which act as a template for the vesicle membrane. The emulsion itself forms the body of the vesicle, while steering of the emulsion using dielectrophoretic force allows the outer leaflet to be "painted" onto the inner leaflet as the emulsion slips through the interface between oil and aqueous phases. Typical emulsion (vesicle) diameters are ~10μm; however, diameters <1μm are achievable with the addition of an electric field to break up larger emulsions. For additional flexibility, the device can be fabricated to allow for the addition of exogenous materials (ie PQS, OMV proteins) prior or subsequent to their formation and also allow for on-chip analysis of the OMVs as they exit. The proposed microfluidic device provides a flexible, reliable and low cost way to produce vesicles to biological specifications at high throughput. These tailored OMVs will then be used to investigate exciting biological questions that were technically unfeasible even in the recent past.

This project is a continuation of research first started as part of the HHMI program in the summer of 2013. Progress has been made in producing the asymmetric vesicles using microfluidic technology. A protocol has been established for building the microfluidic devices and vesicles at the higher end of the required size range have been generated (i.e. diameters > 10 µm). Additional engineering challenges remain, in particular improvements to reliability and throughput of the instrument and the production of sub-micron sized asymmetric vesicles. Full characterization of the vesicles is also required using confocal microscopy and transmission electron microscopy.

Academic preparation for undergraduate students

One student in biology will participate in the project by purifying natural OMVs, investigating the effects of PQS on cell-sized and nanoscale artificial OMVs, and observing the effects of both on biofilm maturation to discriminate between the signaling and physical effects of PQS on bacterial physiology. Ideally the student will have background in biology and some biochemistry. One student in engineering will participate in the project through microfabrication of the microfluidic device and synthesis of artificial vesicles of varying size and composition. Ideally the student will have a background in fluid mechanics and interfacial phenomena.

References

1.             Allesen-Holm M., K. B. Barken, L. Yang, M. Klausen, J. S. Webb, S. Kjelleberg, S. Molin, M. Givskov, and T. Tolker-Nielsen. 2006. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Microbiol. 59:1114–28.

2.             Chiarot P. R., S. I. Gubarenko, R. Ben Mrad, and P. Sullivan. 2008. Application of an Equilibrium Model for an Electrified Fluid Interface—Electrospray Using a PDMS Microfluidic Device. J. Microelectromech. S. 17:1362–1375.

3.             Hatzakis N. S., V. K. Bhatia, J. Larsen, K. L. Madsen, P.-Y. Bolinger, A. H. Kunding, J. Castillo, U. Gether, P. Hedegård, and D. Stamou. 2009. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat. Chem. Biol. 5:835–41.

4.             Mashburn L. M., and M. Whiteley. 2005. Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 437:422–425.

5.             Schertzer J. W., and M. Whiteley. 2012. A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle. mBio 3:e00297–11.

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8. Investigating the Influence of Features, Intensity and Viewpoint on Classification of Expressions in Children or Adult

Faculty Mentors:

Dr. Peter Gerhardstein , Psychology and Dr. Lijun Yin, Computer Science

Graduate Student Mentors:

Xing Zhang, Computer Science and Daniel Hipp, Psychology

This is a joint project in collaboration of Psychology (Prof. Peter Gerhardstein) and Computer Science (Prof. Lijun Yin) to work on the issue of recognizing human facial expressions from both human perception and machine recognition perspectives.

The proposed research will investigate children/adult's ability to classify emotional expressions, using sophisticated 3D image software and a procedure that conceals most of the face, allowing only a small amount to be viewed at any one time. This will reveal the areas that children/adult find most useful in recognizing emotions. This question is important because certain childhood disorders such as autism and other mental health problems (depression, schizophrenia) are marked by an inability (or reduced ability) to recognize emotional expressions.

Humans are able to recognize facial expressions of emotion from faces displaying a large set of confounding variables, including age, gender, ethnicity and other factors. The goal of this project is to apply highly sophisticated 3D model derived stimuli and a recently developed masking technique to the investigation of emotion expression recognition in a developmental framework. The use of such stimuli is relatively new, and there appears to be almost no work applying the masking technique in developmental investigations. The proposed research will increase understanding of children/adults' recognition mechanism for human facial expressions, potentially leading to an increased understanding of developmental recognition, suggestions for improvement of the widely used Facial Action Coding System, and potential insights into clinical issues (autism, depression, schizophrenia, social anxiety disorder) marked by a deficit in recognition of emotional expressions.

Projects and academic preparation for undergraduate students:

- A computer science/engineering student will be involved in designing algorithms for 3D imaging data collection, processing, analysis, and recognition. The student will extend the existing work to develop software for 3D model visualization and image manipulation. The student will also implement the computer program in order to meet the experimental requirement from the collaborating student for conducting human perception experiments. Finally, the algorithm evaluation will also be carried out through machine based expression analysis, model quality assessment and usability study.

- A psychology student will be involved in designing and conducting experiments for following tasks: (1) Exploring differences in loci of information important for expression classification between young children and adults. A relatively new and powerful masking method from the adult vision research literature could be used to test controlled images of facial expressions generated using high-resolution 3D photography and face modeling software; (2) Exploring the impact of changes to model pose on expression classification. Unlike the traditional tests of expression using frontal poses, this project will use the 3D modeling software to generate changes in pose and test classification across development. Activities will also involve interactions with students of computer science in helping on data collection and data analysis.

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9. Sequence Selective Recognition and Imaging of Double Stranded RNA via Triplex Forming PNA

Faculty Mentors:

Dr. Eriks Rozners, Chemistry, Dr. Christof Grewer, Chemistry and Dr. Dennis McGee, Biology

Graduate Student Mentor:

Dziyana Hnedzko, Chemistry

Non-coding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Selective binding, detection and imaging of regulatory RNAs, such as microRNAs, would be highly useful for fundamental biology and practical applications in biotechnology and medicine. However, most non-coding RNAs fold in double helical conformations and molecular recognition of such structures is a formidable problem. Designing small molecules that selectively recognize RNA using hydrophobic or electrostatic interactions has been a challenging process. On the other hand, hydrogen bond mediated base pairing, which is the key feature of nucleic acids, has been underutilized in molecular recognition of RNA. This presents a gap in both fundamental knowledge and practical techniques that limit progress in biochemistry and molecular biology. Since the nucleobases of RNA are already base paired in the double helix, the most selective and straightforward sequence readout would be triple helix formation in the major groove of RNA. However, RNA triple helices have been little studied.

Image of modified hetercycles
Figure 2. PNA uptake by confocal laser microscopy.

pna uptake

The present proposal follows up on PI's recent discovery that peptide nucleic acid (PNA) forms highly stable and sequence selective triple helices with double stranded RNA at physiologically relevant conditions and will test the hypothesis that modified nucleobases (Figure 1) will allow sequence selective recognition of a vide variety of biologically relevant RNAs. The objectives of this project are to: 1) study the triple helical recognition of double stranded RNA using nucleobase-modified PNA (Figure 1); 2) in collaboration with Profs. Grewer (Chemistry) and McGee (Biological Sciences) study the RNA binding and cellular uptake of PNA conjugated to cationic peptides (Figure 2) and 3) in collaboration with Profs. Grewer and McGee develop new fluorescent PNA nucleobases for detection of non-coding RNAs in live cells.

Exploring the sequence selective recognition of RNA duplex will advance fundamental knowledge on molecular recognition of nucleic acids. In contrast to extensive research on DNA triple helices, RNA has received relatively little attention. Surprisingly, before the preliminary studies presented herein there were no reports on stable RNA triple helices using PNA as the third strand. This represents a significant gap in molecular recognition of RNA. Development of sequence selective RNA binders is important for understanding the biochemistry of non-coding RNAs and may find broad applications ranging from fundamental research in RNA biology to analytical detection of regulatory RNAs and even development of novel therapeutic approaches. Preliminary results suggest that PNA is an unexpectedly well-suited ligand for recognition of biologically important RNA species. Development of new fluorescent PNA nucleobases may open doors to new techniques for RNA imaging in live cells.

The PI has long-standing interest and a proven track record in mentoring students. Undergraduate students and PI have co-authored twelve publications in high impact peer reviewed journals. In the present project, students will be able to participate in all three objectives and receive training in an interdisciplinary area spanning chemical synthesis, molecular recognition, biophysics and cell biochemistry.

Academic preparation for undergraduate students:

- A chemistry sophomore or junior who has completed Organic Chemistry Sequence (CHM 232, 332, 335). Introductory Biology and Biochemistry courses will be a big plus but not required. The chemistry student will be focusing on using synthetic organic chemistry and biophysical techniques to synthesize and characterize the chemically modified PNA derivatives.

- A biochemistry or biology sophomore undergraduate student who has completed Introductory Biology (BIOL 117, 118) and Chemistry (CHEM 107, 108). Some Introductory Organic Chemistry is a plus. The biochemistry/biology student will be focusing on preparing cell culture, exposing cells to PNA and characterizing cellular uptake of the chemically modified PNA derivatives.

Mentoring arrangements:

- Dziyana Hnedzko, a graduate student in the Organic Chemistry Laboratory (Prof. E. Rozners) in the Department of Chemistry will mentor the chemistry student.

- TBA, graduate student in the Biological and Biophysical Chemistry Laboratory (Prof. C. Grewer) in the Department of Chemistry will mentor the biochemistry/biology student.

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10. Determination of Glutamate Transporter Involvement in Ethanol Withdrawal related Neuronal Excitotoxicity

Faculty Mentors:

Dr. Christof Grewer, Chemistry; Dr. David Werner, Psychology

Graduate Student Mentors:

Rose Tanui, Chemistry; Jessica Santerre, Psychology

This HHMI project stems from the collaboration between Dr. Christof Grewer and Dr. David Werner aimed at further assessing the functional importance of glutamate transporters in human health and pathophysiology.

Stroke is characterized by ischemia that results in irreversible neuronal cell death and is the third leading cause of death in the United States. Despite alcohol's (ethanol's) widespread use, binging and chronic consumption are associated with increased risk for stroke and likely represent a major factor for future susceptibility. In fact, ethanol withdrawal periods are of particular interest as this vulnerable time is manifested by neuronal hyperexcitability paralleled by increases in glutamatergic receptor upregulation and stimulation that are similar to ischemic models. Unfortunately, neuroprotective measures have yet to be assessed during this vulnerable period. Glutamate transporters are a major player in maintaining normal extracellular glutamate levels, but also contribute to pathophysiological increases in extracellular glutamate excitotoxicity. Therefore, the focus of this project is to better understand the involvement of glutamate transporters in excitotoxictiy and neurodegeneration and to begin to identify whether glutamate transporters represent a viable pharmacotherapeutic target.

Students involved in this research will learn how to generate and maintain primary cortical neurons in vitro. Neurons will be exposed to defined concentrations of ethanol for pre-determined lengths of time followed by glutamate deprivation to mimic ischemia. Glutamate transporter-selective agents will then be assessed to determine their protective efficacy. Neuronal viability and cell death will be measured using cytochemistry, while glutamate elecrophysiological function will be assessed using whole cell recordings. Students will gain skills related to cell biology as well as pharmacology and toxicology in Dr. Werner's lab. Students in Dr. Grewer's lab will gain experience in the synthesis and characterization of novel glutamate transporter selective membrane permeable compounds as well as single-cell electrophysiological analysis. Our combined interdisciplinary may ultimately provide key insights into alleviating the detrimental effects of alcohol as well as potential therapeutic strategies to mitigate ischemic severity related to glutamate dysregulation.

Projects and academic preparation for undergraduate students:

  • Integrated neuroscience students (up to 2) will carry out the neuronal cell culture, ethanol exposure/withdrawal experiments related to glutamate transporter function and cell viability. Such experiments will require the completion of Psyc 362 (Neurophysiology) Psyc 243 (Statistics). Psych 330 (Drugs and Behavior) as well as cell biology and biochemistry are recommended.
  • A chemistry or biochemistry student will assist with the synthesis and characterization of glutamate transporter-related agents and electrophysiological analyses. It is recommended that the successful student will have completed Chem 335 (Organic Chemistry lab) and the related introductory organic chemistry lecture courses.

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11. Development of a Nanodelivery System for Enhanced Treatment of Biofilm-related Infections

Faculty Mentors:

Dr. Amber Doiron, Assistant Professor, Bioengineering and Dr. Karin Sauer, Professor, Biology

Graduate Student Mentors:

Yizhong Liu, Bioengineering and James Goodwine, Biology

Surface-associated bacterial communities known as biofilms pose significant problems in medicine due to their resistance to killing by antimicrobial agents, rendering conventional antimicrobial treatment strategies ineffective. Recent evidence suggests that biofilms formed by Pseudomonas aeruginosa to antimicrobials can be rendered susceptible to antimicrobial agents upon the enzymatic depletion of a metabolite previously demonstrated to not be required for bacterial growth but only for biofilm formation and the maintenance of the biofilm structure. Our primary objective is to develop a nanodevice for the delivery of the metabolite-depleting enzyme to biofilms and ultimately, in vivo biofilm infections.

The goal of the nanoparticle formulation is to encapsulate and protect the stability of the metabolite-depleting enzyme and antibiotics (e.g. tobramycin), to deliver the two concurrently for most efficient biofilm disaggregation and killing, and to provide the basis for a potential future pharmaceutical product for the elimination of biofilms in wounds, with the overall goal of developing a nanodelivery device that is more stable and easier to handle but equally capable compared to free metabolite-depleting enzyme in treating biofilm infections. The nanodevice will be composed of biodegradable, slow release nanoparticles. Nanoparticles will be evaluated for important efficacy-determining characteristics like size, surface charge, release rate, and lack of toxicity to normal human cells. The efficacy of the nanodevice will be tested on Pseudomonas aeruginosa biofilms for their ability to disaggregate biofilms and enhance the killing efficacy of tobramycin compared to free metabolite-depleting enzyme. Additionally, confocal microscopy will be used to determine whether the nanoparticles are capable of penetrating into the biofilm structure.

Academic preparation for undergraduate students:

One student with a bioengineering or chemistry background with interest in drug delivery and biomaterials will contribute to this project. The student will use polymer-based biomaterials to create nanoparticles encapsulating the metabolite-depleting enzyme and antibiotics. The student will then evaluate those particles on the basis of size, shape, surface charge, biocompatibility with normal human cells, and release pharmacokinetics. Ideally, this student will have completed courses in chemistry and biology.

One student with a biology background and interest in microbial biofilms and/or biochemistry will participate in the research project. The student will use microbiological approaches to grow bacteria including aseptic techniques and viability determination assays, as well as various other microbiological and biochemical techniques. Ideally, this student will have completed the course in Microbiology.

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12. The Effect of Bacteria on the Structural Failure of Bronchial Epithelial Cells.

Faculty Mentors:

Dr. Guy German, Bioengineering and Dr. Claudia Marques, Biology

Graduate Student Mentors:

Paul Woods, Biology and Xue Liu, Bioengineering

This BU-HHMI research project brings together the bioengineer Guy German and the microbiologist Cláudia N. H. Marques to research the causes of the failure of eukaryotic cell integrity upon bacterial infection.

Infections of the human body are primarily caused by the presence of bacteria that live as multicellular biofilm communities. As the first step in the infection process, bacterial pathogens need to bind with eukaryotic cell adhesion molecules, such as integrins and cadherins. These molecules however play a vital structural role in tissue formation by enabling intercellular adhesions. Currently, there is a critical lack of knowledge of the effects of bacterial binding on tissue mechanical properties and integrity. While symptoms and methods of fighting bacterial infections have been studied in great detail, very little is understood about the physical mechanics and subsequent changes in heterogeneous structural integrity of epithelial tissues upon infection with bacterial pathogens. The goal of the proposed work will be to establish the heterogeneous mechanical response of eukaryotic cell monolayers to bacterial infection. The binding of bacteria to cell adhesion molecules is expected to disintegrate cell-cell adhesions, causing cells to bind more to the substrate than the surrounding cells. The research will quantify changes in the spatially resolved local and collective mechanical stresses imposed by cells monolayers on an underlying adherent deformable elastomer substrate using traction force microscopy (TFM). The primary relevance of this research is to reveal and to improve our understanding of the fundamental mechanical origins of structural failure in biological tissues.

The experimental procedure will involve a number of stages. The substrates will first be fabricated using simple spin coating techniques. Eukaryotic cells will then be cultured on each substrate in order to form a confluent cell monolayer. Once established, bacterial infection will be initiated and the cells imaged using transmitted light microscopy. Measurements of spatially resolved traction stresses imposed by the cells on the deformable substrate will be achieved by imaging a layer of fluorescent beads embedded within the substrate using fluorescent microscopy. The positions of these beads will be tracked over time and used to quantify spatially resolved interfacial traction stresses imposed by the cells on the substrate. Results from this work are expected to provide a better understanding of the impact of bacterial infection on the breakup of intercellular adhesion and changes in collective cellular mechanics. We hope this research may also reveal potential means of preventing tissue damage and methods of improving infection resolution.

Life Science Student: This student will be involved in seeding the bronchial epithelial cell lines and in performing the cell infection with Staphylococcus aureus and Pseudomonas aeruginosa. The student will also be responsible for parts of the microscopy and for the determination of the virulence factors involved in the structural failure of the eukaryotic cells.

Requires Biol 118. Preferred if completed Biol 224 or Biol 314 or 311.

Physical Science, Math, Computer Science or Engineering Student: This student will be involved in the fabrication of the deformable substrates including fluorescent bead embedding for use in measuring the interfacial cellular traction forces. The student will also be responsible for parts of the microscopy and the subsequent image analysis and quantification of cellular traction forces.

Requires Math 221, Math 222, Phys 131, Phys 132. Preferred if completed BE 318 Biomechanics.

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13. Food Additives, the Gut Microbiome and Metabolic Disorders

Faculty Mentors
Dr. Anthony Fiumera, Associate Professor, Biology and Dr. Gretchen Mahler, Assistant Professor, Bioengineering

Graduate Student Mentors
Zhongyuan Guo, Bioengineering

This HHMI research project brings together a biologist, Anthony Fiumera, and a bioengineer, Gretchen Mahler, to investigate the role of titanium dioxide nanoparticles, a common food additive, on the gut microbiome, intestinal function and the development of metabolic disorders.

Project Proposal
Metabolic disorders are pressing health related challenges. Approximately 35% of American adults and 17% of children are clinically obese(1). Worldwide, obesity rates are projected to nearly double from 2005 to 2015(2). Obese individuals are at an increased risk for a variety of other health conditions including type 2 diabetes, dyslipidemia, hypertension, and coronary heart disease(3), and obesity was estimated to have increased overall healthcare costs in the United States by $147 billion in 2008 alone(4).

Poor diets and sedentary lifestyles are arguably the largest contributors to metabolic disorders(3), but other factors also contribute. Although hundreds of genes have been identified that impact obesity(5), the known genetic variants explain only a small proportion of the observed heritability suggesting a role for genotype by environment interactions. Recent studies have observed associations between the gut microbiome and metabolic disorders(6-11). Factors such as age(12), diet(9), therapeutic drugs(13), and physiological state(11, 14, 15) are known to affect the gut microbiome and may be one of the important links driving the interactions between genotype and environment. As such, any environmental factor affecting the gut microbiome is a potential factor influencing metabolic disorders.

Nanoparticles may be an environmental factor contributing to metabolic disorders through changes to the gut microbiome. Nanoparticles, commonly defined as materials less than 100 nanometers, can occur naturally (e.g. in dust), be produced unintentionally (e.g. in diesel exhaust) or be deliberately engineered for a wide variety of healthcare, commercial and/or industrial applications(16). Ingestion of engineered nanoparticles is nearly unavoidable as they have entered the food chain in products such as agricultural chemicals, processed foods, and nutritional supplements(17). Currently, it is estimated that the average American consumes 1012-1014 engineered fine to ultrafine particles per day(18), mainly TiO2, silicates, and aluminosilicates used as whitening, brightening, and anti-caking agents in processed foods. The amplified surface reactivity of nanoparticles may cause interactions with biological molecules such as DNA, proteins, and cell membranes(19) and their diverse properties make general assessments of their toxicity difficult (20). In vitro bacterial cultures have been shown to be sensitive to nanomaterials(21-29) but the few studies to determine the effects of nanoparticles on the gut microbiome are inconsistent(30),(31).

tio2The long-term goal of this work is to develop and utilize in vivo and in vitro systems to study nanoparticle toxicity, genetic susceptibility, the role of the gut microbiome and the molecular mechanisms underlying genotype-by-environment interactions affecting metabolic disorders. The objective of this proposal is begin developing the in vivo and in vitro systems that can ultimately be utilized as tools to study the effects of nanoparticles on metabolic disorders.

1. Develop an in vitro tool for nanoparticle-biological screening assays (supervised my Dr. Mahler).
Background and motivation: The small intestine is the primary site of nutrient absorption(32), and the role of the gut microbiome on small intestinal function is not well understood or easily studied in vivo. The predominant bacterial species present in the small intestine include lactics, enterics, enterococci, and bifidobacteria(33), and the type and number of bacterial species present may change the way that nutrients are absorbed and lead to conditions that promote metabolic disorders. We have developed a model of the GI tract that has been optimized for investigating nutrient absorption and includes digestion; a mucus layer; and multiple, physiologically relevant cell types(34-36). Our long-term goal is to incorporate a mock community of small intestinal bacterial species into this model to determine how ingested materials change microbial viability, microbial behavior and/or intestinal function. This innovative in vitro method will enable precise control of both the internal cell architecture and external microenvironment, allowing us to rapidly screen for physiologically relevant ingested compound-GI function interactions.

1a. Introduce a single bacterial species into our in vitro GI tract model and determine the effects on intestinal function. We will introduce Lactobacillus rhamnosus GG (L. rhamnosus), a known beneficial bacteria(37), into out GI tract model and use well-established assays to determine how this bacteria influences small intestinal barrier function, nutrient transporter (DcytB, DMT1, ferroportin, and ZnT1) gene and protein expression, and brush border enzyme (aminopeptidase-1, ATPase, sodium-glucose cotransporter, and sucrase isomaltase) activity.

1b. Determine the role of TiO2 nanoparticle consumption on bacterial viability and intestinal glucose transport. We will expose our in vitro GI tract model incorporating commensal bacteria (see Figure 1) to TiO2 nanoparticle concentrations relevant to real-life exposures (108 and 1010 30 nm particles/cm2). We will then measure L. rhamnosus viability, glucose transport (using 2-NBDG, a fluorescently-labeled deoxyglucose analog), and sodium-glucose cotransporter activity.

2. Characterize the effects of nanoparticle exposure on the gut microbiome and phenotypes related to metabolic disorders in the in vivo genetic system, Drosophila melanogaster (supervised by Dr. Fiumera).
Background and motivation: D. melanogaster can be a model toxicology system to study genetic and environmental contributions to changes to the gut microbiome and susceptibility to metabolic disorders Many of the major metabolic homeostasis pathways (e.g. insulin signaling; TOR pathway) are well conserved between humans and flies(38, 39) and Drosophila's relatively simple gut microbiome (four or five common families) has been shown to influence metabolic homeostasis(40-42).

2a. Characterize changes in gut microbiome composition after TiO2 feeding using 16S ribosomal sequencing. Flies will be reared on either control food or food containing relevant concentrations TiO2 nanoparticles (5 and 50 ppm). Adult flies will be flash frozen and the midguts dissected and used for DNA extractions.  The 16S region will be amplified(42), cloned into E. coli and sequenced. Hundreds of clones will be sequenced from each treatment and used to estimate diversity of the microbiome and comparisons will be made with rarefaction curves.

2b. Characterize phenotypic indicators of metabolic disorders and measures of fitness.  We will also use standard assays(42) to investigate protein, triglyceride and carbohydrate levels and we will measure indicators of fitness including male mating success and female fertility(43) in the three treatments.

Academic preparation for undergraduate students
One student in bioengineering will participate in the project by developing and characterizing the GI tract model. Ideally, the student will have BIOL 117-118 and BE 203 completed. One student in biology will perform fruit fly studies and biochemical assays. This student should have completed BIOL 117-118.

References

1.    Ogden CL, Carroll MD, Kit BK, & Flegal KM (2012) Prevalence of obesity in the United States, 2009-2010. NCHS data brief. 82.
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5.    Walley AJ, Asher JE, & Froguel P (2009) The genetic contribution to non-syndromic human obesity. Nat Rev Genet 10(7):431-442.
6.    Le Chatelier E, et al. (2013) Richness of human gut microbiome correlates with metabolic markers. Nature 500(7464):541-+.
7.    Cotillard A, et al. (2013) Dietary intervention impact on gut microbial gene richness. Nature 500(7464):585-588.
8.    Ley RE, et al. (2005) Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences of the United States of America 102(31):11070-11075.
9.    Ley RE, Turnbaugh PJ, Klein S, & Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444(7122):1022-1023.
10.    Turnbaugh PJ, et al. (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444(7122):1027-1131.
11.    Wen L, et al. (2008) Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 455(7216):1109-1113.
12.    Koenig JE, et al. (2011) Succession of microbial consortia in the developing infant gut microbiome. Proceedings of the National Academy of Sciences 108(Supplement 1):4578-4585.
13.    Li M, et al. (2008) Symbiotic gut microbes modulate human metabolic phenotypes. Proceedings of the National Academy of Sciences 105(6):2117-2122.
14.    Koren O, et al. (2012) Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy. Cell 150(3):470-480.
15.    Koren O, et al. (2011) Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proceedings of the National Academy of Sciences 108(Supplement 1):4592-4598.
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18.    Lomer MCE, Thompson RPH, & Powell JJ (2002) Fine and ultrafine particles of the diet: Influence on the mucosal immune response and association with Crohn's disease. P Nutr Soc 61(1):123-130.
19.    Xia T, Li N, & Nel AE (2009) Potential health impact of nanoparticles. Annu. Rev. Public Health 30:137-150.
20.    Maynard AD (2006) Nanotechnology: assessing the risks. Nano Today 1(2):22-33.
21.    Chen H, et al. (2013) Broad-Spectrum Antibacterial Activity of Carbon Nanotubes to Human Gut Bacteria. Small 9(16):2735-2746.
22.    Raghupathi KR, Koodali RT, & Manna AC (2011) Size-Dependent Bacterial Growth Inhibition and Mechanism of Antibacterial Activity of Zinc Oxide Nanoparticles. Langmuir 27(7):4020-4028.
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25.    Diggikar R, et al. (2013) Silver-decorated orthorhombic nanotubes of lithium vanadium oxide: an impeder of bacterial growth and biofilm. Appl Microbiol Biotechnol 97(18):8283-8290.
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29.    Hashim M, et al. (2013) Investigation of structural, dielectric, magnetic and antibacterial activity of Cu–Cd–Ni–FeO4 nanoparticles. Journal of Magnetism and Magnetic Materials 341(0):148-157.
30.    Hadrup N, et al. (2012) Subacute oral toxicity investigation of nanoparticulate and ionic silver in rats. Archives of Toxicology 86(4):543-551.
31.    Fondevila M, Herrer R, Casallas MC, Abecia L, & Ducha JJ (2009) Silver nanoparticles as a potential antimicrobial additive for weaned pigs. Animal Feed Science and Technology 150(3–4):259-269.
32.    DeSesso JM & Jacobson CF (2001) Anatomical and physiological parameters affecting gastrointestinal absorption in humans and rats. Food Chem. Toxicol. 39(3):209-228.
33.    Sekirov I, Russell SL, Antunes LCM, & Finlay BB (2010) Gut Microbiota in Health and Disease. Physiological Reviews 90(3):859-904.
34.    Mahler GJ, Shuler ML, & Glahn RP (2009) Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. The Journal of Nutritional Biochemistry 20(7):494.
35.    Mahler GJ, Esch MB, Glahn RP, & Shuler ML (2009) Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity. Biotechnology and Bioengineering 104(1):193.
36.    Mahler GJ, et al. (2012) Oral exposure to polystyrene nanoparticles affects iron absorption. Nat Nano 7(4):264-270.
37.    Gibson GR (1999) Dietary Modulation of the Human Gut Microflora Using the Prebiotics Oligofructose and Inulin. The Journal of Nutrition 129(7):1438S-1441s.
38.    Leopold P & Perrimon N (2007) Drosophila and the genetics of the internal milieu. Nature 450(7167):186-188.
39.    Baker KD & Thummel CS (2007) Diabetic larvae and obese flies - Emerging studies of metabolism in Drosophila. Cell Metab 6(4):257-266.
40.    Storelli G, et al. (2011) Lactobacillus plantarum Promotes Drosophila Systemic Growth by Modulating Hormonal Signals through TOR-Dependent Nutrient Sensing. Cell Metab 14(3):403-414.
41.    Shin SC, et al. (2011) Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling. Science 334(6056):670-674.
42.    Ridley EV, Wong ACN, Westmiller S, & Douglas AE (2012) Impact of the Resident Microbiota on the Nutritional Phenotype of Drosophila melanogaster. PloS one 7(5).
43.    Vogel A (2013) Effects of atrazine exposure on male reproductive performance in Drosophila melanogaster. MS (Binghamton University).

 

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14. Investigating the Role of Shear Stress in Biofilm Development Using Microfluidic Technology

Faculty Mentors: Dr. Jeffrey Schertzer, Assistant Professor, Biological Sciences

Dr. Paul Chiarot, Assistant Professor, Mechanical Engineering

Graduate Student Mentor: Nicholas Brown, Mechanical Engineering

This HHMI research project brings together a biologist, Jeffrey Schertzer, and an engineer, Paul Chiarot, to develop microfluidic flowcells to be used in biofilm research. The size of these devices will allow for small-volume experiments using limited reagents. The flexibility of the flowcell design will enable investigation into the role of extracellular matrix components in mitigating shear stress during biofilm development.

Flow CellBiofilms are defined as structured, surface-attached communities encased in a complex, self-produced matrix of extracellular polymeric substances (EPS). This mode of growth is predominant in nature1 and is a feature of many bacterial infections2. Growth in a biofilm protects a bacterium from grazing amoeba in natural settings, biocides in industrial settings, potent antimicrobial agents in clinical settings and elements of the immune system while inside a host2. Understanding how these communities are established and maintained is therefore relevant in nearly every context important to microbiologists. Biofilm maturation is a developmental process comprised of several defined steps, involving the differential expression of a large number of genes and the involvement of small molecule signals. Critical to the development of mature biofilms is the assembly of a multicomponent amalgam known as the extracellular matrix. The composition of the matrix has received considerable investigation in several species and is generally considered to consist of polysaccharides, protein filaments and extracellular DNA (eDNA)3,4. Despite extensive investigation into matrix composition, one area that has not been well studied is the contribution of lipids and membrane structures/particulates to the assembly and maintenance of the biofilm. Electron microscopic evidence has confirmed that Outer Membrane Vesicles (OMVs) are abundant in the biofilm matrix of several species, including the model organism Pseudomonas aeruginosa5. Our own work has confirmed that the ability to produce OMVs is important for proper maturation of biofilm architecture (Fig 1b vs. 1c). Interestingly, preliminary results suggest that this phenomenon may be a function of the shear stress experienced by the growing biofilm. We hypothesize that some matrix components may be dispensable under conditions of low shear, but that the combinatorial effects of a full complement of matrix components allows the biofilm to withstand the high shear associated with rapid flow and/or narrow growth environments. This will be investigated by generating mutant strains of P. aeruginosa incapable of synthesizing or exporting specific matrix components and analyzing their ability to develop mature biofilms. In addition, eDNA can be specifically removed by the exogenous addition of nuclease to the growth medium, and OMV production can be modulated through the presence or absence of exogenously added PQS (a small molecule stimulator of OMV production). Use of either of these valuable tools requires the development of small-volume biofilm reactors (flowcells) to minimize reagent consumption. In addition, the ideal flowcell will be transparent and accessible enough to allow easy access of microscope objective lenses for the characterization of biofilm architecture. To achieve these goals, we will make use of microfluidics technology.

The fabrication and use of polymeric microfluidic devices is central to this project for building the bacterial flowcells. Leveraging microfluidic technology has many important advantages. These include: exceptional control over the applied shear stress and improved optical access for observing the biofilms. The devices will be built using layers of poly-dimethylsiloxane (PDMS) bonded to a glass substrate. Soft- and photo- lithographic methods are used to build the microfluidic devices. Our team has extensive experience with microfabrication and we have already established the process for building these devices. The process involves casting PDMS on to a negative mold of a microchannel network. The molds are built using photolithographic patterning of the photoresist SU8 that is spin-coated on to a silicon wafer. The cast PDMS forms half of the channel network. An enclosed network is formed by irreversibly bonding it under oxygen plasma to a rigid (glass) substrate. All of the equipment used to build the microfluidic chips is available to the faculty mentors at SUNY Binghamton. The benefits of this method for building flowcells are its low-cost and flexibility. Any design of a microchannel network can be built by modifying the mold. The low-cost means the chips are disposable. The completed microfluidic devices (i.e. flowcells) are fitted into a custom-built fixture that facilitates external fluid connections (Fig 1a). The fixture consists of two pieces; a recessed lower plate that the microfluidic device sits in and an upper plate that holds the device in place. Biofilms that grow on the glass surface inside the microchannel network can be easily observed (Fig 1a). Openings machined in the fixture permit the use of brightfield and confocal microscopy for precise characterization of biofilm structure under varying experimental conditions.

flow mediaThe biofilm will experience a shear stress as the growth medium flows over its surface (Fig 2a). Inside a microfluidic device, the magnitude of the applied stress can be precisely controlled. Two parameters are available to tune the stress: the growth medium flow rate and the microchannel geometry. Modulating the microchannel geometry is the preferred method of control so that the total volume of media delivered to any flowcell can remain constant. In addition, when the channel cross-section is non-uniform, the shear stress applied to the biofilm will vary along the flow direction. This means different magnitudes of stress can be applied to the same biofilm inside a single microchannel (this would not be possible by modulating the flow rate alone). It is possible to create virtually any microchannel geometry using our proposed method for building microfluidic devices; including straight channels, converging/diverging channels, curved channels, etc. (Fig 2b). The appropriate geometry will be selected based on the required shear stress profile. The exact magnitude of the stress applied to the biofilm can be calculated since the media flow rate and channel geometry are known. For a uniform channel with a rectangular cross-section (4mm x 1mm) and media flow rate of 1 ml/hr, the shear stress at the center of the channel is approximately 1 milliPascal (Fig 2c; red line). However, the exact stress can be calculated anywhere inside the microchannel. Determining the stress for a non-uniform cross-section is more complicated and may require the use of numerical modeling; for example, using the commercial computational finite element package ANSYS Fluent.

Academic preparation for undergraduate students

One student in biology will participate in the project by generating mutant strains of P. aeruginosa, cultivating biofilms and analyzing their architecture by both light and confocal laser scanning microscopy. In addition, the student will assist in developing quantitative metrics to describe various states of biofilm development. Ideally the student will have background in biology and some biochemistry. One student in engineering will participate in the project through the design and fabrication of the microfluidic devices. They will also use analytical and computational methods to characterize the fluid flow. Ideally the student will have a background in fluid mechanics.

References

(1)            Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappin-scott, H. M. Microbial biofilms. Annu Rev Microbiol 1995, 711–745.

(2)            Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Bioflims : A Common Cause of Persistent Infections. Science (80-. ). 1999, 284, 1318–1322.

(3)            Flemming, H.-C.; Wingender, J. The biofilm matrix. Nat Rev Microbiol 2010, 8, 623–633.

(4)            Karatan, E.; Watnick, P. Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Mol Biol Rev 2009, 73, 310–347.

(5)            Schooling, S. R.; Beveridge, T. J. Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol 2006, 188, 5945–57.

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15. Human Traffic Impacts on Microbiological Metal Cycling in Roadside Soils and on Biodiversity in Urban Streams.

Faculty Mentors: Prof Weixing Zhu, Biology and Prof Thomas Kulp, Geology

Graduate Mentors: Matt Lundquist, Biology and Meghan Dovick, Geology

This HHMI research project brings together ecologist Weixing Zhu and geo-microbiologist Thomas Kulp, who have been working on several projects relating human impacts, particularly through traffic, on environmental geomicrobiology and biodiversity in urban ecosystems. The projects chosen for this collaboration involve studies on roadside ecosystems that are in part funded by the Wallace Research Foundation (a continuation of our roadside ecosystem project), and a new urban stream project.

Roadside ecosystems (areas adjacent to roadways including many urban streams) are exposed to traffic related pollution including elevated inputs of reactive forms of nitrogen (N: mainly NH3 and NOx), metals (e.g., Zn, Cu, Cd, Sb), and road salt (as winter deicing agents). Microbiologically mediated oxidative and reductive transformations directly affect the solubility, mobility and toxicity of toxic metals in natural settings. Salinity is an important environmental factor in controlling the makeup of microbiological communities in soils and other ecosystems, and the energetic costs of coping with life at high salinity may limit the types of microbial metabolisms that may be employed in a given setting. Microbiological cycling of metals also represents a critical component of the global biogeochemical cycles of carbon, nitrogen, and sulfur. The salinity effects on microbiological cycling of metals in roadside soils, however, have not been well studied.   In addition, metal, salt, and nitrogen pollution associated with vehicular traffic and roadways may alter the biodiversity of benthic organisms in urban streams, often chosen as indicator species in human-impacted ecosystems.

Students will conduct metal analyses (Zn, Cu, Cd, Sb, and other selected metals) in the Geology laboratories. They will use microwave digestion to separate metals from the soil matrix and then analyze metal concentrations using the ICP-OES and ICP-MS. In the Biology laboratories, they will sample benthic invertebrates from area streams exposed to various urban impacts. Samples of organisms will be analyzed for nitrogen and phosphorus contents, as well as metal concentrations. Microbiological microcosm experiments will be conducted with roadside soil material collected from varying distances to the roadway. The potential for soil microbial populations to oxidize various toxic metals of interest, and associated rates of oxidation, will be assessed in microcosms prepared over a range of total salinity concentrations.

Academic Preparation

We are looking for two motivated Biology/Geology/Environmental Studies sophomore or junior undergraduate students for this interdisciplinary biogeochemistry project on roadside ecosystems. Desirable course background for the Biology student includes general Ecology (Bio 355) and Chemistry I and II, while the Geology student should have completed Environmental Hydrology (342) and either Environmental Studies 201 or Earth Surface Processes (Geol 211). The two students will work together on elucidating the biogeochemical coupling of C, N, and metals and the effects of varying salinity on the observed processes, as well as exploring the causes and mechanisms of biodiversity change.

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Last Updated: 6/19/14