Annelise E. Barron - US grants

9870386 Barron This POWRE proposal is from a very new educator and one who could profit substantially from an award. the research involves the synthesis and evaluation of a novel class of peptides where the normal alkyl chain attached to the alpha-carbon is instead attached to the amide nitrogen. The synthesis of these amino acids is easier than conventional amino acids, a broader range of these glycine derivitives is possible, and the resultant polymers are more resistant to enzymatic hydrolysis. The primary emphasis will be on developing folding rules for these natural protein mimics. The ease of synthesis will facilitate the study. The new class of polypeptoids has interesting potential applications as biomaterials. ***

Cutting-edge DNA sequencing technologies rely upon the use of miniaturized electrophoresis channels microfabricated on chips or provided by large arrays of fused-silica capillaries. Microchannel electrophoresis systems are easily automated, allow the use of large voltage gradients for rapid DNA separations, and consume very little sample. With these advantages, miniaturized systems have the clear potential to deliver both the order-of-magnitude increase in sequencing throughput and substantial decrease in the cost per base that are called for by Human Genome Project. However, the full speed-increase and cost-saving potentials of miniaturized sequencing systems will only be realized with the development of a new class of replaceable DNA sequencing gels that will provide both long read lengths and rapid, low-pressure, microchannel loading, characteristics that are mutually exclusive in media formulated from conventional water-soluble polymers. Here, we present results clearly demonstrating the potential of thermo-responsive polymers to decouple the loading and sieving properties of microchannel sequencing gels, by simultaneously providing the high-resolution, long-read-length DNA separations typically expected from high-viscosity solutions of high- molecular-weight polymers, and the rapid, low-pressure loading usually considered possible only with low-viscosity, low-molecular-weight-polymer solutions. This superposition of desirable gel properties is achieved with polymers designed and synthesized in our laboratories to have a thermally-controlled viscosity switch. Using acrylamide-based formulations, we will develop 'thermo- melting' gels enabling rapid, low-pressure loading at 65 C and high-resolution sequencing at 45 C. We will also develop 'thermo-gelling' formulations for application in high-temperature sequencing -- gel that will be easily loaded at 25 C, and can be run at 75-85 C. High-temperature runs eliminate sequence-dependent compression zones occurring in repetitive regions of genomic DNA. Finally, we aim to develop advanced gel formulations with thermally-tunable meshes, to push back the limits in sequencing read lengths. The sequencing and loading performance of these novel gels, designed specifically for microchannel electrophoresis, will be tested both in capillaries and on microchips.

0093806
Barron
A current frontier in bioengineering is the design of non-natural, sequence-controlled polymers that can effectively mimic some of the structures and activities of natural proteins, but that are non-immunogenic and stable as in vivo therapeutics. This exploratory research requires an integration of principles and paradigms taken from biochemistry and biophysics with methods of biochemical and biomedical engineering. A focused, medically-relevant research project is proposed that, even if only partially successful, will have far-reaching, fundamental implications for the new field of biomimetic polymer engineering,

An important and tractable problem is chosen: The criticalneed for improved synthetic analogs of the human surfactant proteins SP-B and SP-C. Preliminary data is shown and new strategies are proposed towards the development of novel SP-mimics based on polypeptoids: non-natural polymers based on a peptide backbone, yet differing in that sidechains are bonded to backbone nitrogens rather than to a-carbons. N-substituted peptoids with proteinogenic sidechains previously have been shown to be extremely protease-resistant, and further to raise only very low-level immune response in vivo. In the investigator's lab and elsewhere, some peptoid sequences have been shown to adopt stable, helical secondary structure in aqueous or organic solution. Sequence-specific peptoids can be synthesized easily, at substantially lower cost than peptides, with facile access to diverse sidechain chemistries.

Natural lung surfactant coats the internal surfaces of mammalian lungs and enables normal breathing. It is a complex mixture composed of 95% lipids and 5% surfactant-specific proteins (SP). Both protein and phospholipid fractions play critical roles in lung surfactant s physiological properties, providing a decrease in the work of breathing by regulating surface tension at the air-liquid interface of alveoli as a function of their surface area. The amphipathic, helical surfactant proteins SP-B and SP-C (79 and 35 amino acids, respectively) promote rapid phospholipid adsorption to the air-liquid interface, facilitate respreading of the phospholipid monolayer throughout the respiration cycle, and regulate the phase behavior of the monolayer to yield the lowest possible alveolar surface tension. The absence or dysfunction of lung surfactant on alveolar surfaces leads to respiratory distress syndrome (RDS) in which lungs are incompliant and vulnerable to collapse. Patients with severe RDS cannot be mechanically ventilated without damage to lung tissue, and require immediate surfactant replacement therapy. Premature infants (< 30 weeks) are born with immature lungs lacking surfactant, and often suffer from severe RDS. These patients typically receive surfactant replacement therapy at birth with an animal-derived surfactant formulation. Synthetic formulations exist, but are significantly less efficacious than natural surfactant in enabling proper lung functioning, primarily because they lack effective functional mimics of SP-B and SP-C. Medicines sourced from animals raise concerns about a possibility for cross-species pathogen transmission and also for immunogenicity. Therefore, the development of an effective synthetic biomimetic surfactant replacement is a present need.

Aims to address this need, as well as to carry out important fundamental research are:

(1) To design, synthesize, purify, and characterize the secondary structure of peptoid-based mimics of the helical, amphipathic lung surfactant proteins SP-B and SP-C. Methods include organic synthesis, analytical and preparative HPLC, mass spectroscopy, and circular dichroism (CD);

(2) To confirm and further characterize the in vitro biophysical functioning of peptoid-based SP-B and SP-C mimics as spreading agents for biomimetic phospholipid admixtures. Experimental approaches include both pulsating bubble surfactometry and Langmuir-Wilhelmy surfactometry.

(3) Based on these carefully-repeated preliminary results, to prepare another proposal to the NSF Directorate of Bioengineering to continue the research project thereafter.

DESCRIPTION: (provided by applicant) NIH-NHGRI support is requested to defray participant costs for a special ACS symposium entitled "Microscale Separations and Analysis," which will focus on the cutting edge of technology development for genomic and proteomic analysis. This one-day symposium (August 27, 2001) will be held in Chicago, IL as part of the 2001 annual meeting of the American Chemical Society. The panel of speakers is highly interdisciplinary and includes both academic and industrial scientists (Annelise Barron, Chemical Engineering, Northwestern; Robert Austin, Physics, Princeton; Andrea Chow, Chemical Engineering, Caliper Technologies; Harold Craighead, Applied and Engineering Physics, Cornell; Jed Harrison, Analytical Chemistry, University of Alberta; Stevan Jovanovich, Mechanical Engineering, Molecular Dynamics; Barry L. Karger, Analytical Chemistry, Northeastern University; Stephen Quake, Applied Physics, Caltech; J. Michael Ramsey, Analytical Chemistry, Oak Ridge National Laboratories; Gary Slater, Physics, University of Ottawa; Jonathon Sweedler, Analytical Chemistry, University of Illinois; Stephen Williams, Analytical Chemistry, ACLARA BioSciences; Edward Yeung, Analytical Chemistry, University of Iowa/Ames Laboratory). In all, the participating scientists include 7 engineers and physicists and 6 analytical chemists, all of whom head research programs focused on development and application of technology for genomic and/or proteomic analysis in capillaries and microfluidic devices. The goal of the symposium is not only to educate the broader chemical sciences community in the exciting challenges of the area, but also to bring together the analytical chemists, who might have attended this meeting anyway, with the physicists and engineers who would have been highly unlikely to attend. This will promote idea exchange and collaborations across disciplines. Three industrial speakers and two scientists from National laboratories are included in the program, to highlight how technology development for the genome centers through collaboration and commercialization. 15% of the speakers are women, reflective of the generally low female representation in this field at the Principal Investigator level. However, given the "star quality" of many of the speakers and the great diversity of ACS attendees, it is hoped that attendance of the symposium will be high and that the event will attract more women and under-represented minority scientists to the field.

We propose to develop a novel class of biomaterials called "polypeptoids," or poly-N-substituted glycines, and to apply them to a specific biomedical problem: the need for more effective synthetic, functional mimics of the human lung surfactant proteins SP-B and SP-C. Lung surfactant (LS) is a surface-active material that coats the internal surfaces of healthy mammalian lungs and enables breathing, by reducing the surface tension on the alveolar surfaces. LS is composed of 95 percent surface-active lipids and 5 percent surfactant-specific proteins; both lipid and protein fractions are necessary for its functioning. Two of these surfactant-specific proteins, SP- B, and SP-C, are especially surface-active and are critical for the proper biophysical functioning of LS in vitro and in vivo. SP-B and SP-C are both small, helical, amphipathic proteins (79 and 35 amino acids, respectively); essentially, just peptides. Premature infants born before about 30 weeks of gestation are born with immature lungs lacking surfactant, and require the delivery of an exogenous lung surfactant replacement at birth to enable mechanical ventilation. At present, the most efficacious LS replacement formulations are animal- derived, and therefore raise concerns about their level of purity, their consistency of formulation, and their potential for pathogen transmission, as do any medicines sourced directly from animals. While synthetic LS replacements do exist, they do not work as well as animal- derived surfactant replacements, primarily because these formulations lack good functional replacements for SP-B and SP-C proteins. We propose to develop functional mimics of SP-B and SP-C based on poly-N-substituted glycines, which are sequence- specific heteropolymers synthesized in a similar manner to synthetic polypeptides, by a facile, automated solid-phase protocol. Peptoids offer the advantage s of protease- resistance, biomimetic helical secondary structure, low immunogenicity, and low cost. Peptoid-based SP-mimics will be synthesized, purified, and their secondary structure and biophysical surface activities will be analyzed in vitro circular dichroism spectroscopy and by equilibrium and dynamic surfactometry. The feasibility of these novel SP- mimics is demonstrated in preliminary work. Promising formulations will be tested in vivo by a collaborator.

The award from the Instrumentation for Materials Research Program in the Division of Materials Research will allow Northwestern University to acquire a new Fourier Transform Infrared Spectrometer (FTIR) Instrument for research and education in the Departments of Materials Science and Engineering, Chemical Engineering, and Chemistry. The proposed FTIR instrument will replace the current outdated equipment and will be housed in the Polymer Characterization Facility of the Materials Science department. The instrument will consist of two modules: a conventional turn-key FTIR spectrometer with an array of attachments for standard spectroscopic techniques, and a highly customizable module specifically designed for measurements of molecular structure in ultra-thin films of organic molecules. The former will make the instrument easily useful to a broad audience of Northwestern researchers, while the latter will allow measurements of molecular conformations in more difficult samples, especially in biomaterials and nanomaterials research. The instrument will facilitate the training of undergraduate and graduate students in all three departments, by being utilized in laboratory sections of polymer materials class offered to the engineering undergraduate students, and a spectroscopy class offered through the chemistry department. The more advanced capabilities of the spectrometer will greatly advance the ability of students involved in the interdisciplinary bio- and nanomaterials research at Northwestern to elucidate and understand the molecular-level details of structure in their samples.
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The award from the Instrumentation for Materials Research Program in the Division of Materials Research will allow Northwestern University to acquire a new Fourier Transform Infrared Spectrometer (FTIR) Instrument for research and education. The instrument will be housed in the Polymer Characterization Facility in the Materials Science and Engineering department, and will be used to characterize new polymeric, biological and nanomaterials prepared by researchers at Northwestern. In particular, it will enable measurements of the orientation, shape, and arrangement of molecules in extremely thin (just a few molecules thick) films deposited on the surfaces of solids and liquids. Such films are often critical in creating the new materials for biotechnology and nanoscience applications, governing, for example, the interactions of biomaterials with biological tissues, or the workings of nanoscale sensors capable of precisely measuring minute quantities of substances present in a gas or a liquid. The instrument will be used in instruction for several classes currently taught to science and engineering students at Northwestern. Moreover, students of the undergraduate and graduate levels will use the instrument for their research, improving the quality and scope of their training as future members of the science and engineering workforce.

DESCRIPTION (provided by applicant): It is proposed to optimize, evaluate, and pilot rapid, scalable, and low-cost microchip electrophoresis technologies for sensitive and specific molecular detection of cancer by tandem single-strand conformational polymorphism (SSCP)/heteroduplex analysis (HA), using the p53 gene as a model system. We request a 1-year R21 phase and a 3-year R33 phase. The proposed project involves collaboration between members of Northwestern's Lurie Comprehensive Cancer Center, including researchers in Chemical Engineering, the Medical School, and Evanston Hospital. Microchannel "tandem" SSCP/HA is a novel mutation detection method recently developed in our laboratory, which involves the simultaneous generation and analysis of homo/heteroduplex DNA and SSCP conformers. Studies of a significant number of samples (32) indicate that tandem SSCP/HA allows for much higher-sensitivity mutation detection (100%) than SSCP alone (93%) or HA alone (75%), for p53 samples. We have developed and published optimized sample preparation protocols, gel formulations, and analysis conditions for capillary array electrophoresis (CAE). During the R21 phase, we will translate these methods to microfluidic electrophoresis chips, which offer a large increase in throughput and drop in cost of DNA analysis compared to CAE. The p53 gene, known to be mutated in >50% of human cancers, and whose mutation status can be predictive of patient response to chemotherapy, is the important model system chosen. However, microchip-based genetic analysis technologies to be developed should be easily applied to ANY cancer-related gene. In the R21 phase, we will analyze approximately 60 different DNA samples derived from tumor cell lines, representing a range of mutations in different p53 exons, to determine the impact of DNA sample characteristics and electrophoresis protocols on the sensitivity and specificity of the method, in a blinded study designed by collaborating biostatisticians. When optimized tandem SSCP/HA protocols have been developed for microchips, they will be piloted by the analysis of >200 selected samples amplified from frozen, solid tumors banked at Evanston Hospital. Via this blinded study, sensitivity and specificity (both expected to be at or near 100%) will be determined and reported for the first time using banked tumor tissue, providing necessary validation for clinical application of this technique, and making rapid, low-cost cancer genotyping technology widely available to physicians.

[unreadable] DESCRIPTION (provided by applicant): Current state-of-the-art DNA sequencing technologies rely on electrophoresis in large arrays of fused silica capillaries, which are filled with entangled polymer matrixes that provide DNA separation. Major cost savings, and a large increase in throughput, would be realized if next-generation sequencers could be based on glass or plastic microfluidic devices. However, microfluidic devices currently under development, like today's capillary array instruments, require viscous polymer matrixes for DNA separation, which inherently limit read length and are difficult to load into chip rnicrochannels. The development of a new and practical paradigm for microchannel DNA sequencing in free solution would greatly increase the chances that microfluidic devices could be made practical for high-throughput DNA sequencing in Genome Centers. Here, we present results that clearly demonstrate the potential of End-Labeled Free Solution Electrophoresis (ELFSE) to sequence DNA in microchannels without a sieving matrix. The basic principle of ELFSE is to attach a monodisperse perturbing entity, or "drag-tag", to each DNA molecule in a sequencing mixture, allowing separation based on DNA size by free-solution rnicrochannel electrophoresis. Novel drag-tags and microchannel wall coatings invented in our laboratory show highly promising properties for DNA sequencing by ELFSE. We have just finished building an advanced chip electrophoresis system. With further development, ELFSE on chips could be the next generation of easily automated, high-throughput, long-read length DNA sequencing technologies. Using a combination of genetic engineering and organic chemistry techniques we will synthesize and purify non-natural, monodisperse drag-tags based on repetitive, nonnatural polypeptides ("protein polymers"). Optimized protocols for conjugation of drag-tags to DNA primers, cycle sequencing, and sample cleanup will be developed. Protocols for DNA sequencing by ELFSE will be developed and optimized using both a 4-color capillary array instrument and microfluidic chips. A variety of wall coatings based on previously reported chemistries would be explored for minimization of interactions between drag-tags and capillary walls to maintain high-efficiency DNA peaks. The theory of ELFSE is under development, and further efforts will be geared to theoretical predictions and molecular dynamics simulations of ELFSE. Theoretical predictions will guide every aspect of ELFSE experimentation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): NIH support is requested to defray participant costs for a special Materials Research Society (MRS) symposium entitled, "Biological and Bio-inspired Materials Assembly," which will focus on the cutting edge of research on novel biomaterials based on DNA, protein, and lipid assemblies. This 3-day symposium will be held in Boston, MA as part of the 2003 Fall Meeting of the Materials Research Society. The panel of invited speakers is highly interdisciplinary and includes a wide array of academic scientists (Angela Belcher, Materials Science & Engineering, Massachusetts Institute of Technology; William DeGrado, Biochemistry & Biophysics, University of Pennsylvania; David Lynn, Chemistry, Emory University; Phillip Messersmith, Biomedical Engineering, Northwestern University; Martin Moller, Biochemistry, RWTH; Christof Niemeyer, Biochemistry, University of Dortmund; Nadrian Seeman, Chemistry, New York University; Samuel I. Stupp, Materials Science and Engineering, Chemistry, and Medicine, Northwestern University; Gregory Tew, Polymer Science & Engineering, University of Massachusetts; Matthew Tirrell, Chemical Engineering, University of California at Santa Barbara; Ulrich Wiesner, Materials Science & Engineering, Cornell University; Shuguang Zhang, Center for Biomedical Engineering, MIT). 12-14 other talks will be chosen from submitted abstracts. The invited speakers include 7 bioengineers and 5 biochemists and chemists, all of whom direct research focused on the development of biomimetic systems for self-assembly of biological and non-biological systems. The goal of this symposium is not only to educate the broader materials research community about the importance and potential of biomaterials research, but also to bring together the materials scientists, who might have attended this meeting anyway, with biochemists, chemical engineers, and chemists, who would have been unlikely to attend. This symposium, and the planned conference dinner associated with it (for the speakers), will promote idea exchange and interdisciplinary collaboration. One speaker is female, reflective of the generally low female representation in this field at the principal investigator level. One of the three organizers is also female. Given the "star quality" of many of the speakers and the diversity of MRS meeting attendees, it is hoped that attendance at the symposium will be high and that the symposium will attract more women and under-represented minority scientists to the field.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests funding continuation for the Predoctoral Biotechnology Training Program at Northwestern University (NU). This interdisciplinary and interdepartmental Program offers biotechnology training opportunities for a select group of Ph.D. students from four participating units: the departments of Biomedical and Chemical Engineering (ChE), the Chemistry department, and the Interdepartmental Biological Sciences (IBiS) graduate program. Research opportunities, chosen from among a wide array of laboratories in the life sciences, are complemented by a core interdisciplinary curriculum. Three- to 6-month industrial internships expose trainees to the industrial environment of modern biotechnology. All trainees and most preceptors participate in the IBiS, two-day, off-campus annual retreat, which allows extensive interactions among students and faculty from many disciplines and increases the cohesiveness of the trainee group. The Biotechnology research club, which meets once a month or more during the academic year and twice a month during the summer, is the forum where trainees and faculty present their research results to and interact with other students participating in the Program. Several other interdisciplinary research clubs (e.g. Biophysics Club, Biomaterials Club) provide trainees with additional opportunities to broaden their knowledge and perspective. Trainees organize and host biotechnology seminars that bring industrial and academic scientists to campus for a seminar and discussions with trainees. Students also take advantage of several other life sciences-related seminar programs on campus. Instruction in the responsible conduct of research is carried out primarily through the course "Ethics in Biological Research". Trainees are recruited by the Program and the participating units, using posters, brochures, and WWW pages. Intensive efforts are made to recruit students from underrepresented groups, and have been quite successful. In the first 5 years of this Training Program a significant fraction of trainees has come from ChE because of the larger student-applicant pool. During the past 4 years, however, a diverse population of graduate students from BME, IBiS, and ChE has been supported in a balanced manner. The number of preceptors participating in the Program will increase from 18 to 31. An increase from 5 to 8 funded positions will support this expanded preceptor base and allow the support of some Chemistry students. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to create and study a new family of biomimetic oligomers for antibacterial applications. In particular, we aim to create biostable, functional mimics of natural antimicrobial peptides (AMPs), which are an integral and effective part of the host-defense systems of myriad organisms ranging from humans to bacteria. By preferentially binding to and disrupting or permeating bacterial cell membranes, these surface- active peptides are able to kill a broad spectrum of microorganisms. Many are also selective, causing no harm to mammalian cells. The killing mechanism, while imperfectly understood, is sufficiently general that bacteria have been unable to evolve resistance to AMPs over millions of years. Thus, good functional mimics of AMPs hold forth the promise of serving as a new class of antibiotic compounds, which could act in solution directly or be tethered to the surfaces of biomedical devices to stave off infection. We propose to create non-natural mimics of AMPs, since peptides themselves are vulnerable to proteolysis and hence degrade too rapidly in the body, and moreover because peptides are often immunogenic in vivo. In particular, our novel mimics will be based on amphipathic, sequence-specific oligo-N-substituted glycines ("peptoids"). Peptoids are quite similar in structure to peptides, yet are protease-resistant. They are easily synthesized by a high-yielding, solid-phase protocol that allows the easy incorporation of biomimetic side chains. Moreover, peptoids can be designed to form stable, biomimetic helices that keep their folded structure in both aqueous and biomembrane environments, and which are highly resistant to denaturation. In preliminary work, we have shown that certain peptoid sequences (9-17mers) designed to mimic the amphipathic sequence patterning and helical structure of antimicrobial peptides are potent and selective antibacterials (MIC ~ 4 [unreadable]M against E. coli and 820 nM against B. subtilis, with negligible hemolysis at the E. coli MIC). We propose to further study and develop these compounds, by (1) exploring structure-function relationships and mechanism(s) of action through detailed biophysical studies of a "basis set" of closely related peptoids with differing potency/selectivity profiles, (2) creating novel peptoids that mimic structural motifs of natural AMPs, including peptoids that are lipidated, cyclized, and kinked;(3) testing their antibiotic activity against antibiotic-resistant bacteria as well as early-stage investigation of their toxicity to human cells.

DESCRIPTION (provided by applicant): The obtainment of a high-quality sequence of the very first human genome has provided us with incredibly important information. However, in order to better understand the origins of genetic diseases such as cancer, sequencing data must be obtained from multiple human genomes and tumor genomes, as well as from the genomes of other complex organisms. To enable this important work, the high costs associated with sequencing the DMA of complex organisms must be greatly reduced. The current workhorse technology used in Genome Sequencing Centers, capillary array electrophoresis, has reached a plateau in terms of the cost per sequenced base, and we request NIH/NHGRI funds to complete our work in the development of novel polymer networks and wall coatings to enable a lower-cost, more efficient sequencing technology to take its place: microfluidic chip electrophoresis. DNA separation is much faster and cheaper in microfluidic chips, because much narrower DNA sample zones are injected. (We demonstrate here the sequencing of 550 bases, with 98.5% base-calling accuracy, in just 5.5 minutes - a new speed record by a factor of three! Capillaries require at ~ 70 minutes to give the same result). Chip-based technology also promises to enable the total integration of the sequencing process: one will merely load sheared BAG DNA on the chip, and the Sanger reaction, sample cleanup, and separation of DNA to give called sequence will be done automatically. We will create and test polymer matrices and wall coatings that enable read lengths of 700 to 800 bases in <10 minutes on glass chips with human genomic samples. Other matrices and coatings will be developed to deliver, at a minimum, 500-base reads on plastic chips (a lower-cost chip substrate) in <15 minutes. Our approach is to carefully control polymer and copolymer synthesis conditions and accurately characterize the physical and chemical properties of the polymers, so that we can better understand and design the next generation of materials for improved performance. The polymer networks we create will be of sufficiently low viscosity to enable automated, low-pressure loading (<150 psi) of matrix into chips and will be 'self-coating'to remove extra steps in the chip preparation process, increasing throughput and lowering costs. We will carefully study the molecular mechanisms responsible for the ultrafast DNA sequencing we have recently observed in our polymer matrices, a critical aspect of optimizing microchip-based sequencing systems.

DESCRIPTION (provided by applicant): DNA sequencing is currently in the midst of disruptive technological shifts, with 454, Illumina, and Solid providing us with enormous throughput increases and large reductions in cost per base. Massively parallel technologies deliver a few Gbp of sequence per week as short fragments, or reads. New applications of sequencing only recently considered impractical are enabled: personal genome sequencing, "metagenomics" analysis of 'soups'containing several, to hundreds of unique organisms, and finally, de novo sequencing of novel genomes of complex organisms. No matter how the sequencing is done, reads must be assembled computationally, if they are to be useful. Given the read length and read quality limitations of new instruments and the massive volume of data generated, the computational assembly problem is becoming critical, with the cost of computational infrastructure and personnel exceeding reagent and instrument-related costs. Moreover, the results of assembly are currently far from ideal;for example, much of the human genome remains invisible due to high percentage of repeats. We propose to develop a new "front end" to next-gen sequencers for DNA preparation, the "Read-Cloud Method", which can reduce computational cost of genome assembly by 2-3 orders of magnitude, produce more complete and accurate genomes, and make metagenomics tractable. We propose a hierarchical sequencing approach, without any need for bacterial cloning. We will achieve this by handling single DNA molecules, tiled across the genome with high redundancy, on microfluidic devices. We will design, prototype, and thoroughly test technology to (i) shear genomic DNA into 200- kbp fragments with narrow size distributions;(ii) randomly amplify each individual, 200-kbp DNA in isolation, within a porous gel microcontainer that will be formed around the dsDNA molecule within a microdevice;(iii) digest micro-encapsulated DNA into small fragments, of tunable size;(iv) bar-code the progeny of each 200-kbp DNA with a 12mer oligonucleotide, to identify each read as associated with a particular 200-kbp DNA. A planar microfluidic device will be fabricated to allow one unique bar- code sequence to be blunt-end-ligated to both DNA termini. Bar-coded DNA is pooled, and next-gen sequencing is done. The results are a highly reducible data set. The method and algorithm are applicable universally, to next-generation platforms. The PIs (Batzoglou, Barron, Shaqfeh, Quake) will collaborate to make an efficient approach to hierarchical sequencing in microfluidic devices. PUBLIC HEALTH RELEVANCE: Project Narrative Gene sequencing is important to medicine. Our DNA sequencing method has the potential for reducing computational cost by orders of magnitude while making the assembled genomes significantly more complete and accurate. The key to this step is using microfluidic handling technologies to subdivide genomic DNA into 200kbp fragments, which are then amplified in isolation from each other and uniquely-labeled to form a highly reducible dataset for genomic assembly.

DESCRIPTION (provided by applicant): Debilitating craniofacial skeletal defects, which occur frequently as a result of congenital disorders, trauma, cancer, and surgical interventions, are some of the most challenging problems for reconstruction. While infants demonstrate the ability to heal large and complex calvarial defects, those older than one year of age have an insufficient healing response to even small skull defects. Although a plethora of strategies have been developed over the past century, the patchwork of methods currently available reflects the inadequacies of each therapeutic technique. In this regard, skeletal stem cell biology holds enormous untapped promise for future tissue engineering applications. The goal of this project is to make autogenous skeletal progenitor cell-based craniofacial skeletal regeneration a clinical reality. Using an interdisciplinary approach, we will bring together our expertise in stem cell biology, bioengineering, and craniofacial surgery to tackle the roadblocks to translation of cell-based skeletal tissue engineering. In Specific Aim 1, we will prospectively isolate pure populations of human skeletal progenitor cells from different tissues and identify key progenitor cell niche factors necessary for their expansion and differentiation. In Specific Aim 2, we will develop a novel high throughput, microfluidics-based FACS (MF-FACS) device for simultaneous isolation and encapsulation of individual skeletal progenitor cells in a hydrogel-based, microenvironment conductive to their regenerative capabilities. In Specific Aim 3, we will assess the regenerative potential of transplanted encapsulated skeletal progenitors in critical-sized calvarial defect models. The role of supplementary niche factors will be further assessed using a tunable macroscale hydrogel scaffolding material. We believe this highly innovative project will ultimately produce an effective cell-based craniofacial skeletal regeneration regiment suitable for clinical testing. PUBLIC HEALTH RELEVANCE: The current tools available to doctors to repair bone structures of the head and face damaged from accidents, birth defects or cancer are inadequate. Using cells that have been removed from the patient, this project seeks to identify, protect, and return only those cells capable of growing into new bone structures. We seek to improve public health by developing a point-of-care method for surgical reconstruction of living bone.

Project Summary/Abstract Increasing prevalence of Alzheimer?s dementia (AD) is a growing health and economic crisis. Although studied for 112+ years, the root causes for sporadic AD?which is > 95% of AD?are unclear. Over the last 15 years, 415+ clinical trials to test new drugs against AD failed. Approved drugs can only manage symptoms. I will use NIH Pioneer funding to investigate a novel hypothesis for the etiology of sporadic Alzheimer?s dementia, based on my insight that imbalance between two innate immune peptides may be a key factor that modulates the risk of formation, the stability, and clearance of AD-associated fibrils and plaques. Recent observations of chronic P. gingivalis and Herpesvirus infections being associated with Alzheimer?s fit this hypothesis. I am, to my knowledge, the only researcher working on this idea. The human cathelicidin LL-37, unique in our proteome, is an antiviral and antibacterial defense peptide deployed by microglia, macrophages, neutrophils, epithelia, B cells, and NK cells (to kill infected cells). Thus LL-37 is a centrally important defense peptide, necessary for killing bacterial and viral pathogens and infected host cells. LL-37?s Vitamin D3-, RXR-agonist-, and butyrate-dependent expression is also stimulated by infection, wounding, exercise, and some vaccines (e.g., BCG & OPV vaccines). Certain pathogens, P. gingivalis in particular, release enzymatic virulence factors that rapidly degrade LL-37. Degradation of LL-37 could well dysregulate the brain?s innate immunity, causing neurodegeneration; in LL-37?s absence, the immune process of macroautophagy is crippled. The Alzheimer?s-associated peptide Ab now seems also to be a host defense peptide; brain infections by either Herpesviridae or P. gingivalis stimulate Ab production, causing it to accumulate in plaques that co-locate with pathogens. Recently I and collaborators showed that LL-37 and Ab are both expressed in human brain, and bind each other sequence-specifically. LL-37/Ab binding prevents fibrillization and blocks Ab from adopting b-type secondary structure. Thus, LL-37 degradation may allow Ab to accumulate. Our in vivo studies show that cathelicidin induction in 5XFAD mice slows AD progression and improves 5XFAD cognition to match wild-type. I aim to tie this finding to infection-associated dementia. In this Pioneer project, I will use wild-type and cathelicidin KO mice to demonstrate that degradation of LL-37 by P. gingivalis virulence factors may well be one cause of brain tissue degradation leading to dementia, which can be prevented by early upregulation of cathelicidin to prevent infection; or treated orally with antimicrobials. My lab has developed new antimicrobials that potently kill both P. gingivalis and inactivate Herpesvirus.