Bacteroides fragilis is an opportunistic pathogen, occurring normally in the human digestive tract, but causing severe and often life-threatening abscess formation and sepsis outside of this environment. There is growing international interest in B. fragilis from several points of view. These include questions regarding its commensal role in the gut during human assimilation of nitrogen, and its pathogenic effects in causing opportunistic infections. Studies on nitrogen assimilation in B. fragilis are fundamental to the understanding of how this organism exists within the gastrointestinal environment and how it out-competes the majority of other gut organisms in becoming the most successful opportunistic pathogen if it breaches the mucosal barrier. Information about how this process occurs could lead to useful strategies for drug design and disease prevention.
This research proposal focuses on two key enzymes, glutamate dehydrogenase and glutamine synthetase, that are involved in nitrogen metabolism in B. fragilis. The research question to be addressed is
The approach used will be to characterise the regulation of these enzymes in response to various nitrogen sources. The research will employ a multidisciplinary approach including genomic, proteomic and structural biology studies.
Rationale and Motivation
Bacteroides fragilis is an anaerobic opportunistic pathogen, occurring normally in the human digestive tract, but causing abscess formation and sepsis outside of this environment. The majority of infections caused by these organisms occur following injury to the gastrointestinal tract and disruption of its mucosal wall. This may occur, for example during abdominal surgery or following a perforated appendix. This allows the bacteria to escape from the gut and to invade tissues in various body sites where they can cause significant tissue damage, anaerobic septicaemia and even death. B. fragilis is associated with approximately 75% of post-operative intra-abdominal wound infections and gangrenous and perforated appendicitis, respectively [cite num=”1″]. South African studies confirm that acute appendicitis has become the commonest non-trauma related abdominal emergency amongst black patients, and that there has been an increase in the number of cases seen due to better diagnosis of this condition among Africans [cite num=2]. Madiba et al. [cite num=3] conducted a retrospective review of appendicitis among 645 African patients at the King Edward VII Hospital in Durban, South Africa and reported a 43% perforation rate complicated by peritonitis, and a 2% mortality rate. In addition, B. fragilis has been reported as a high risk factor in elderly patients on continuous ambulatory peritoneal dialysis [cite num=4], and has also been implicated in causing brain abscesses in infants [cite num=5]. These complications increase the need for costly antibiotic therapies and prolong patient hospitalization times.
Role of bacterial nitrogen assimilatory enzymes in nitrogen assimilation in B. fragilis
Nitrogen assimilatory enzymes play a significant role in B. fragilis metabolism, both in the gut and during the infective process. The Bacteroides, functioning as commensals, make an important contribution to the utilisation of various nitrogenous foods that enter the large intestine, particularly in the degradation of proteins [cite num=6] [cite num=7]. The levels of enzyme activity are dependent on growth conditions both with regard to the nitrogen source (organic vs inorganic nitrogen) as well as the nitrogen concentration. Proteases, which act as virulence factors during pathogenic tissue invasion, increase in activity in response to nitrogen limitation and ammonia starvation [cite num=8] [cite num=9]. They also act as virulence factors, and aid the spread of the organism through tissues by degrading structural proteins and other substances involved in local infection containment. B. fragilis has high protease and peptidase activities [cite num=10], which in turn provides the substrates for further nitrogen assimilatory enzymes, described below. B. fragilis cannot use amino acids as the sole source of nitrogen and relies on the presence of ammonia or peptides to meet its nitrogen requirements [cite num=11] [cite num=12]. Several lines of evidence have led to the hypothesis that the regulation of nitrogen assimilation in this organism occurs via a novel mechanism.
Glutamate dehydrogenase (GDHB)
In most other microorganisms, the glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes function in concert to assimilate ammonia when nitrogen is limiting and only synthesise glutamate dehydrogenase (GDH) under excess nitrogen conditions. B. fragilis cells, however, possess appreciable levels of GDH activity irrespective of whether they are grown with an excess of organic nitrogen or limiting quantities of ammonia [cite num=12][cite num=13]. This phenomenon has been found to be due to the fact that B. fragilis has two GDH enzymes that represent the primary pathway of nitrogen assimilation in this organism. GDHA (dual NADPH / NADH co-factor specific) is induced by low ammonia and low peptide conditions, while GDHB (NADH co-factor specific) is expressed only under high peptide conditions and is not regulated at all by ammonia levels. The GDH enzymes occupy a pivotal role within central metabolism by providing a link between carbon and nitrogen metabolism and may be related to virulence [cite num=14].
We have cloned and characterised the B. fragilis gdhB gene that codes for the NADH specific GDHB enzyme. The protein is cell-surface associated and is regulated by peptides at both the levels of transcription and post-translation [cite num=15] [cite num=16]. We have purified the enzyme both from the recombinant clone expressed in E. coli [cite num=16] as well as in its native form from B. fragilis (Christine Lee, unpublished results). Currently, nothing is known of the mechanisms by means of which the regulation of GDHB at both the transcription and translation levels is achieved, or the role of aminopeptidases and proteases produced by the organism in generating peptides to mediate regulation in the gut and during tissue invasion. Furthermore, the existing structures of homologous enzymes from Clostridium symbiosum and various Archea do not explain the co-factor preference of the B. fragilis enzyme or its possible role in catabolism of glutamate to generate alpha ketoglutatarate for the TCA cycle. These questions will be addressed in this study.
Glutamine synthetase (GS)
The B. fragilis GS enzyme provides an essential route for cells to acquire glutamine under conditions of ammonia starvation, since exogenous amino acid cannot be taken up. This enzyme also has novel genetic and structural features.
The gene encoding the B. fragilis GS (glnA) was previously cloned and characterized in our laboratory, and the recombinant GS protein over-expressed and purified from E. coli. These studies showed that glnA encodes a novel GS (GSIII) that differs in DNA sequence, and subunit size and, possibly, arrangement from those found in many other prokaryotes [cite num=17]. We have done structural alignment of GSIII and S. typhimurium GS (unpublished results). This shows that one intersubunit contact is lost and that the structure of the ADP binding pocket is dramatically altered by an insertion of approximately 40 amino acids. This is in addition to the C terminal and N terminal extensions.
After the discovery of the GSIII in B. fragilis, it was subsequently also reported to be found in B. thetaiotaomicron, Butyrivibrio fibrisolvens, and Ruminococcus spp, an indication that this form of GS might be a feature of anaerobic micro-organisms. Searches of the NCBI database showed, however, that it also has significant amino acid similarity to the glnN protein of Synechococcus spp (a photosynthetic blue-green alga), as well as the glnA protein of Deinococcus radiodurans. It is particularly intriguing to find homologues of the GSIII occurring in such evolutionarily distant organisms as obligate anaerobes, a photosynthetic, free-living blue-green alga, and a highly radiation resistant aerobe. Further, analysis of nitrogen assimilation in such “phylogenetically distinct” organisms may reveal interesting evolutionary links. Studies on nitrogen regulation in Synechococcus have shown that glnN is mediated by NtcA, a member of the Crp/Fnr family of transcriptional activators [cite num=18]. NtcA positively regulates the expression of several genes involved in the assimilation of alternative nitrogen sources under conditions of ammonia limitation including, amongst others, the glnN coding for GSIII [cite num=19].
Currently, very little is known about the regulation of the B. fragilis glnA gene, or the protein structure/function relationships of any of the GSIII group of GS enzymes. While the GSI and GSII enzymes that occur in most prokaryotes and eukaryotes respectively, have been crystallized and their structures solved by electron microscopy and X-ray diffraction, no work at all of this kind has been done on the novel GSIII protein. GSI and GSII have been shown to be complex multi-subunit (12 subunits) allosteric enzymes [cite num=20]. Since our previous work showed that GSIII is substantially different from the other GS groups (possibly 6 larger subunits), no reliable conclusions can be reached by homology modeling concerning its tertiary and quaternary structure, and would render prediction of possible drug targets impossible. Structural studies are, therefore, essential. We are in a particularly strong position to be the first research group internationally to do this (see facilities and expertise of investigators later).
Summary of significance of the work
An understanding of the structure-functional relationships of the GS and the GDHB enzymes in particular would inform future possible antibacterial drug targeting strategies against a medically important anaerobe which is resistant to most current drug therapies other than metronidazole. In addition, an analysis of the molecular and structural features of two of the key nitrogen metabolic enzymes would provide unique insights into the evolutionary development of a primitive anaerobe.
Relevance of the work to Focus Area
- The project offers the opportunity for cutting edge research in two related disciplines to come together to develop new models regarding the structure, function and regulation of novel systems in a medically important organism. The methods to be used mark the first time that structural analysis of this high level will be done in South Africa.
- It will develop the knowledge base both in molecular genetics and structural biology. This will provide a platform for future work in the area of targeted drug development that could be applied to other related research questions in other organisms of medical and industrial importance.
The work will contribute to the development of sound concepts at the level of basic science by posing fundamental questions and using appropriate scientific approaches to answer them.
- Both the field of study and the multidisciplinary approach will ensure that there will be both national and international interest in the work, especially since it will be of an exceptionally high research standard as evidenced by the track record of the investigators, research facilities and calibre of research students involved (see below).
- The project is being submitted to the NRF in this focus area following feedback from the MRC that this type of basic research is not in line with their clinical research emphasis.
- The proposed project is feasible and meaningful, and will be the first one of its kind in South Africa.
- The project proposal offers a multi-disciplinary approach to the study by bringing together genomic and structural biology researchers in creating a knowledge base through addressing common areas of interest from a variety of perspectives.
Although B. fragilis plays a significant role in both the colonic environment and disease processes, fundamental aspects of its physiology remain poorly understood. This is particularly so with regard to nitrogen assimilation and its regulation. The potential involvement of proteases and the assimilation of peptides in the pathogenicity of these organisms highlight the need for further analysis of these pathways.
The objectives of this research are:
- To identify the physiological conditions controlling the activity of key nitrogen assimilatory enzymes (glutamine synthetase and glutamate dehydrogenase) in B. fragilis,
- To characterise the genetic loci adjacent to the genes encoding these enzymes,
- To determine the regulatory mechanism modulating their activities in this organism, and
- To determine the structure of GDHB using electron microscopy and X-ray diffraction, and relate this to its functions.
- To determine the structure of GSIII using electron microscopy and relate this to its functions.
Research Design and Framework
The project consists of two subprojects, each of which contains genetic as well as structural biology approaches. Each aspect of the project will be conducted by a designated student, and all aspects will run simultaneously over three years.
Sub Project 1: Regulation and structure of glutamine synthetase
Molecular genetics studies.
Our preliminary studies have shown that the B. fragilis glnA gene has features of promoter structure that resemble that of the NtcA-related promoters. We have used the (as yet not annotated) B. fragilis whole genome sequence generated by the Wellcome Trust Sanger Centre (http://www.sanger.ac.uk/Projects/B_fragilis/), to identify a possible ntcA homologue. It occurs immediately downstream of the glnA gene and may play a similar role as a global nitrogen regulator in B. fragilis.
We will use PCR techniques to clone this ntcA homologue from B. fragilis, and confirm the cloning by sequence analysis.
We will test the involvement of NtcA in nitrogen metabolism by testing for its transcriptional regulation by nitrogen. B. fragilis will be grown under various nitrogen conditions and mRNA transcription of this gene will be monitored using northern hybridisation (slot blots) of transcripts to a labelled ntcA DNA probe.
We will test the functional similarity of the gene and its product to NtcA by doing complementation studies in a suitable Synechococcus ntcA mutant strain.
We will construct a series of promoter fragments fused to a xylosidase reporter gene for further analysis of ntcA expression in B. fragilis in response to various nitrogen sources.
We will over-express the NtcA protein using a protein expression vector and confirm that it does, in fact, bind to the predicted region of the glnA promoter. This will be done by means of gel binding studies and DNA footprinting analysis.
We will use a proteomics approach to conduct a full analysis of the global induction or repression of B. fragilis of proteins following growth in various nitrogen sources. This will done using 2-D electrophoresis. Discussions are currently underway to do this aspect of the work in collaboration with Prof Mark Morrison in his laboratory at the University of Illinois, USA.
Students involved: 1 MSc student (with upgrade to PhD)
Supervisor: Dr VR Abratt
Structural Biology studies
No structural analysis has been done worldwide on the GSIII group of proteins. Our sequence analysis has shown that the enzyme has 230 additional amino acids relative to the crystallographically determined GSI Salmonella typhimurium enzyme. We aim to use the B. fragilis enzyme as a paradigm for the GSIII enzyme group.
The recombinant B. fragilis GSIII protein will be purified from E. coli carrying the glnA gene on a multicopy plasmid. Biochemical characterization of the purified GS will be done and MALDI TOF used to confirm that the protein is GSIII.
Three-dimensional cryo-EM techniques will be use to determine the number of subunits, their quaternary arrangement and shape of the molecular envelope.
We will interpret the molecular envelope by fitting a model based on the homologous region of GSI.
The work described above will form the basis of further X-ray crystallographic characterization of the enzyme which will be initiated in 2007 and does not form part of the current proposal.
Students involved: 2 MSc (Biochemistry and Structural Biology) possible extension to PhD.
Supervisors: Prof T. Sewell (EM studies and modeling), Dr C. Kenyon (Purification of the protein and biochemical characterization)
Sub-project 2: Regulation and structure of glutamate dehydrogenase B (GDHB).
Molecular genetic studies
Analysis of the DNA flanking regions of the cloned gdhB gene have shown the presence of two truncated open reading frames (ORFs) which may also be involved in nitrogen metabolism and be linked to GDHB activity. The upstream truncated ORF is a putative ptsP gene homologue. The ptsP genes of certain bacteria encode the EINtr enzymes that transport nitrogen-containing compounds into the cell. Downstream of gdhB is a putative aminopeptidase gene homologue that could contribute to B. fragilis protease activity as well as serving to provide the peptides that regulate gdhB expression.
We will use the B. fragilis whole genome sequence of the Wellcome Trust Sanger Centre (http://www.sanger.ac.uk/Projects/B_fragilis/) to generate PCR primers specific to the putative downstream aminopeptidase, as well as the upstream ptsP genes.
Both these genes will be cloned and their sequence confirmed.
The putative aminopeptidase will then be functionally analysed with respect to peptide substrate enzymatic specificity.
Transcriptional (mRNA) induction of the putative amino peptidase and the nitrogen transport gene (ptsP) in B. fragilis grown on a variety of peptide substrates will be analysed using northern hybridisation to confirm whether they are regulated in response to nitrogen.
The effect of a range of synthetic peptides on gdhB induction will be analysed by growing B. fragilis containing a gdhB promoter fusion reporter gene construct.
Comparisons will be made between the range of peptides generated by the aminopeptidase and those that are able to induce gdhB expression.
Students: 1 MSc student with possible upgrade to PhD
Supervisor: Dr VR Abratt
Structural Biology studies
The GDHB protein has NADH co-factor specificity. Yet in preliminary experiments to test for phosphorylation of the enzyme, we have found that the co-factor specificity shifts to being NADPH-dependent after phosphokinase treatment. Model-building has failed to explain this phenomenon. We plan to use x-ray crystallographic structural analysis to resolve this unusual observation.
The recombinant B. fragilis GDHB protein will be purified from an E. coli clone carrying the gdhB gene on a multicopy plasmid. The protein will be examined using EM techniques, bioinformatics and protein modelling to determine its subunit structure.
The GDHB protein will be crystallised and examined using X-ray crystallographic techniques. The structure will be solved by molecular replacement using Clostridium symbiosum GDH as a model.
The cofactor-binding site will be examined under various conditions and in the presence and absence of cofactors and substrate. One specific goal is to determine the reason for GDHB’s failure to accept NADPH as a cofactor
Students involved: 2 MSc (Structural Biology) with possible upgrade to a PhD.
Supervisors: Prof T. Sewell
Availability of specialised equipment, infrastructure and resources
Our laboratories are particularly well placed to conduct this interdisciplinary study. The PI, Dr Abratt at the University of cape Town, has specialised anaerobic growth facilities available, and a proven track record of research in the areas of anaerobic microbiology and molecular genetics, particularly of the Bacteroides nitrogen metabolism.
In addition, we have established the capability to determine protein structure in Africa for the first time through a grant by the Carnegie Corporation of New York to B.T. Sewell (co-investigator). This grant has enabled the creation of the joint UCT/UWC Masters programme in Structural Biology and has, in particular led to the establishment of a facility for protein X-ray crystallography at the University of the Western Cape and the establishment of a protein NMR facility at the University of Stellenbosch. Six masters students have been recruited to the programme in its first year (2003). Each student cohort will take two years, including a year of coursework and a year of research. Three cohorts will be partially sponsored by the Carnegie Corporation grant. Two lecturers will be appointed, Dr Muhammed Sayed, a protein crystallographer who has recently completed a post-doctoral fellowship at the University of Cambridge and Dr Arvind Varsani who has recently completed his PhD at the University of Cape Town and who has experience in molecular biology, electron microscopy and computer modeling of proteins. The programme also calls on seven internationally renowned structural biologists who act as consultants and who assist in both teaching and project work. The programme has been located within the Institute for Infectious Diseases and Molecular Medicine at UCT and the Department of Biotechnology at UWC with the specific intention of contributing to the creation of a resource for drug discovery and the development of industrial enzymes.
The Principle Investigator (PI) heads and supervises her own research group consisting of 7 postgraduate students (Honours, MSc and PhD levels) and is an established researcher with a proven track record in the field of the molecular genetics of nitrogen metabolism of B. fragilis. Students working on the current proposal will join this team, and we will therefore build on and exploit the strengths and advantages of our current research.
The Co-Investigator is the only researcher trained in protein x-ray crystallography currently working in South Africa. This expertise will be utilised in the proposed project. Recent collaborations with British and American researchers have re-established his active contributions to structural biology and have led to his leadership role in the establishment of the joint UCT/UWC Structural Biology Program. His key recent successes include the determination of the structures of several cyanide degrading nitrilases and the E461K mutant of GroEL. These structures have been presented at several local and international conferences and two of the nitrilase structures have been submitted for publication. One of these has been accepted for publication in Applied and Environmental Microbiology.
The PI has published her previous work in this field in internationally well-rated peer-reviewed scientific journals, and also presents her research on an on-going basis at local and international conferences. These include oral presentations at the Anaerobe 2000 Conference in Manchester, UK, and the Anaerobe 2002 Conference in Park City, Utah, USA, June 2002. These conferences are sponsored jointly by the Anaerobe Society of the Americas and the Anaerobe Society of Japan, and bring together the top international researchers in the field of the Microbiology of anaerobes. Because of its importance as a pathogen, a whole session is devoted to Bacteroides fragilis.
Envisaged measurable outputs
Knowledge of the regulation and active sites of these two key enzymes (GSIII and GDHB) can potentially guide the development of antibacterial strategies targeted specifically at eliminating opportunistic infections caused by B. fragilis.
In addition postgraduate students will be trained in a variety of cutting-edge molecular and structural biological techniques. The results of the research will be presented at international conferences in the fields of medicine, microbiology and structural biology. The research and approach are of great interest to the research communities and will be published in international peer reviewed journals.
Progress to date
Considerable progress has been made in providing insights into the mechanisms of nitrogen regulation in B. fragilis, as evidenced by the conference presentations and peer-reviewed journal articles published by us in this field (see sections”Rationale and Motivation” and “Workplan” above). We are now in a very strong position to move forward rapidly. We should do so without delay given the international interest in this organism and the Wellcome Trust”s decision to fund sequencing of the B. fragilis genome. The availability of the genome database provides genomic tools with which to gain a much broader view of the field, as do our discussions with Prof Mark Morrison (USA) in looking at global nitrogen regulation using a proteomics approach.
With regard to the structural biology aspects of this proposal, major progress has been made over the past year. We have run a pilot project to determine the feasibility of the proposal as described in the workplan. The following outcomes have been achieved:
- We have over-expressed the GDHB protein on a high copy number vector in E. coli.
- We have purified the protein to the homogeneity required for crystallization using NADH-specific affinity chromatography.
- The protein yield is sufficient for the needs of the proposed crystallization procedures.
- We have used a matrix approach to crystallizing the protein. This included variations in buffer composition, cations and concentrations.
- The matrix approach resulted in the growth of three different crystal forms.shown in the Appendix 1.
- These crystals are currently being examined using the Rigaku RUH3R/Raxis4++ x-ray diffractometer.
- An MSc program in Structural Biology is being run for the first time this year as a joint UCT – UWC degree. One of the students on this program (Mr Jason van Rooyen, who holds a Prestigious Bursary from the NRF) participated in the GDHB protein purification and crystallization work described above and will continue the structural biology aspects of this work.
Potential Impact on HR Development
The proposed project offers the opportunity for at least 6 post-graduate students to be trained in a range of research techniques as part of a multidisciplinary team. The proposed collaboration with Prof Morrison offers an opportunity for students to be trained in a cutting edge technique not currently available at our institution. As well as participating in the laboratory-based activities, the students will be involved in research related discussions, oral presentations and in the writing of research reports and papers. Computer based skills will be incorporated into all aspects of the research and presentation activities.
In addition, the co-investigator on this project, Prof Sewell, is the convenor of a new MSc course in Structural Biology that began in February 2003. This course is being run as a collaboration between the Universities of the Western Cape and Cape Town and aims to equip students with skills in this rapidly growing and novel scientific area. Structural Biology is an essential tool for the development of biotechnology, which has been identified as a priority area for South Africa. Structural biology is critical to research areas such as rational drug design and the design of enzymes for industrial processes. This new MSc programme in Structural Biology aims to provide graduate scientists and engineers with the broad based knowledge of biological systems needed for this multidisciplinary field. Students specialising in this field, as well as by students specialising in molecular biology, will be involved in the projects described above. Indeed the existence of the Programme provides opportunities never before available in South Africa for doing the work described above.
The staff development grant is required to fund Dr Brendon Price, currently not NRF rated and employed as a Chief technical officer in the Electron Microscope Unit (UCT). He is also actively involved in the protein purification and crystallization aspects of the project. It is anticipated that through working on the project and through attending appropriate courses, he will become an independent protein crystallographer.
Dr Abratt (PI) and Prof Sewell (CI) have been active supervisors of post-graduate students of all races and both genders as is reflected in the list of past and present students below.
Dr Abratt (PI):
Numbers of students graduated MSc and PhD:
13 BSc (Hons) (2 black males, 2 black females, 8 white females, 1 white male 2 MSc (black males)
3 PhD (1 black male, 1 Taiwanese female, 1 white female)
1 PhD candidates to graduate in June 2002 (1 black male).
Current numbers of postgraduate students under supervision:
1 Post doctoral fellow (black male)
4 PhD students (1 black female, 3 white female)
2 BSc Hons (2 white women)
Undergraduate students currently involved in laboratory research projects
3 (3rd year) Microbiology students (2 black female, 1 white female)
Prof BT Sewell (CI)
Postgraduate Students Graduated
2 PhD (white male)
2 MSc (1 white female, 1 white male)
7 MSc (3 white male, 1 white female, 3 black male)
1 PhD (black male)
Potential Impact on Redress & Equity
Both the Principal Investigator and Co-investigator have been active in the science outreach areas highlighted below in a sustained way. They bring their research work to the fore, wherever possible, during these activities in an attempt to engage the scientific and general communities in the scientific process and its value to society. This research area on Bacteroides fragilis provides an ideal vehicle for this approach since it has great scientific interest as well as public relevance to the health and well-being of all South Africans through the possible development of improved drug treatment strategies.
Dr VR Abratt
Co-organiser of the UCT Microbiology · Department exhibit for MRC/EBG Schools’ Science Day (1997) and YEAST focus week in the Western Province (1998) ·
South African Women in Science and Engineering ·
Projects promoting the role of Women in Science and Engineering
Training of HDE students in gender awareness in the school science classroom.
Molecular and Cell Biology Department
schools’ liaison responsibilities (Workshadow program)
Science Day co-ordinator (2000, 2001, 2002)
Board member – ACTIV SCIENCE, a UCT student initiative· aiming to tutor high school students in Maths and Science.
Annual participation in the UCT J.O.B program, an equity driven program to provide vacation employment and training for selected Microbiology and Biochemistry students.
UCT SciFest committee
Workshop coordinator, UCT exhibit, SciFest 2000, 2001, 2002 Grahamstown.
Prof BT. Sewell
Developed Computer programs to help FET level students to learn science. (http://www.physchem.co.za)
Chair of Board of the College in the FET sector aimed specifically at the advancement of historically disadvantaged learners.
Participated in YEAST (1998).
Knowledge of the regulation and active sites of these two key enzymes (GSIII and GDHB) can potentially guide the development of antibacterial strategies targeted specifically at eliminating opportunistic infections caused by B. fragilis.
The project will generate the development of highly trained research students with scarce skills who will be able to apply their minds and scientific acumen to the many areas of research and biotechnology of strategic importance to South Africa. Based on our past record of research in this area, we anticipate continued international interest in this field in the form of international conference presentations and the publication of articles in peer reviewed scientific journals.
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