Huilin Li, Ph.D. - US grants

DESCRIPTION (provided by applicant): We request a 4K by 4K CCD camera designed for cryo-EM. The camera will be installed in a JEOL JEM2010 FasTEM/STEM microscope. The cryo-EM facility is dedicated to studying the 3D structures of biological macromolecules. These protein complexes play critical roles in human health and disease. The microscope is located in Brookhaven National Lab and operated by Huilin Li, who holds a joint appointment between BNL and Biochemistry and Cell Biology Department in Stony Brook University. The large format camera and its associated software will enable microscopy automation and cryo-TEM and cryo-STEM bimodal tomography, thus significantly increase the microscopy throughput and research productivity. There are three large institutes on Long Island, New York, that conduct NIH-supported research. These are the so-called Long Island Tri-Institutes: SUNY Stony Brook being in the middle, Brookhaven National Laboratory about 15 miles to the east, and Cold Spring Harbor Laboratory about 15 miles to the west. There are numerous needs for cryo-EM imaging of large biological assemblies from local researchers. However, our cryo-EM group is the only facility available on Long Island. The requested instrument will facilitate existing NIH-funded cryo-EM projects, with the likelihood of accommodating several more projects from local biomedical community. Public Health Relevance: We request a CCD camera designed for recording digital images in an electron microscope. The microscopy facility is dedicated to studying the 3D structures of biological macromolecules. These protein complexes play critical roles in human health and disease. The large format camera and its associated software will enable microscopy automation and significantly increase the microscopy throughput and research productivity.

DESCRIPTION (provided by applicant): Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents off-pathway interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of virulence-associated surface structures by a broad range of pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. The goals of this proposal are to probe the structure and function of the usher to gain an understanding of the molecular mechanisms governing pilus biogenesis across the outer membrane and to use the chaperone/usher pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the hypothesis that the usher functions as a dimeric complex in the outer membrane to recognize, recruit and position chaperone-subunit complexes to catalyze the exchange of chaperone-subunit for subunit-subunit interactions, promote ordered pilus assembly, and allow secretion of the pilus fiber to the cell surface. The first specific aim will investigate the function of the usher as a catalytic machine and gated secretion channel for pilus biogenesis at the bacterial outer membrane. The second specific will determine the structural basis for pilus assembly and secretion at the outer membrane usher. This application will provide insights into mechanisms of protein secretion, organelle biogenesis, and the assembly of bacterial virulence factors.

DESCRIPTION (provided by applicant): Secondary Analysis of Longitudinal Trait in Genome Wide Association Studies Abstract: Relating genome wide association study (GWAS) data to longitudinal phenotype data can provide special advantages, but also represents certain challenges. This is particularly challenging in secondary data analyses from case-control studies, as commonly found for GWAS investigations. Secondary data analyses using existing case-control GWAS data yield an effective and practical solution for genetic investigations of longitudinal traits that afford the opportunity to examine disease heterogeneity over time and early disease detection. None of the current secondary methodologies works with longitudinal data. The primary goal of this proposal is to investigate and develop statistical inference methods to analyze longitudinal secondary traits using case-control GWAS data. This project is motivated by research problems arising from the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO), in which Cancer Antigen 125 (CA125) and Prostate Specific Antigen (PSA) levels were measured at baseline (T0) and then annually for five years in women and men, respectively, and BMI values were measured or reported at three time points (age 20, 50 and enrollment). The Total Genotype Set developed recently at the NCI for GWAS, includes genotype data for subjects from various case-control cancer studies within PLCO. This aggregated genotype dataset provides a unique opportunity to evaluate the inherited determinants of these serum antigens and their serial time trajectories, as secondary analyses. Our specific aims are: 1) Develop statistical approaches for secondary analysis of longitudinal traits in GWAS. We propose to integrate the mixed-effects model, commonly used for longitudinal analysis, with weighted likelihood and retrospective likelihood methods, two theoretically justified secondary analysis methods for a single time trait, to develop robust and efficient approaches for secondary data analysis of longitudinal traits. 2) Translate the proposed statistical methodology into practical research knowledge and software. We will apply the proposed methods generated in Aim 1 to the PLCO BMI, CA125 and PSA GWAS data to evaluate the inherited determinants of these antigens and their serial time trajectories. We will also develop, distribute and support freely available software packages for the methods used in this proposal. Considering the immense amount of recently generated genotype data, there is a tremendous need for the development of sophisticated secondary analysis statistical methods, as proposed herein. This study will greatly advance applied epidemiology research, by allowing investigators to incorporate longitudinal data analysis with genetic analysis, taking advantage of the large supply of recently generated case-control GWAS data.

? DESCRIPTION (provided by applicant): Initiation of the eukaryotic chromosome replication is a tightly regulated multi-step process. Failure to ensure once and once only initiation event per cell division cycle can result in uncontrolled cell proliferation and tumorigenesis. Central to the replication initiation is the origin recognition complex (ORC) that recognizes the chromosomal origins of replication. With the help of replication initiator Cdc6 and replication licensing facto Cdt1, ORC recruits and loads the Mcm2-7 helicase onto DNA, forming a highly stable Mcm2-7 double hexamer. This completes the first milestone - formation of pre-replication complex (pre-RC). A series of subsequent events culminate to formation of pre-initiation complex (pre-IC) and leading cell to enter into the S phase. X-ray crystal structures have been reported for key proteins involved in prokaryotic replication initiation, but little is known about the structures o the corresponding eukaryotic complexes. This is so because the eukaryotic replication initiators assemble into large and transient complexes, which have been exceedingly difficult for crystallography. Recent advance in hardware and software has made cryo-EM an ideal approach to studying these complexes at much improved resolution. We therefore propose to study how the pre- Replication Complex is established by cryo-EM. This work will advance our knowledge in the initiation of eukaryotic chromosome replication.

DESCRIPTION (provided by applicant): (must be no longer than 30 lines of text) Cryo-EM is a powerful tool for visualizing molecular and cellular structures. Stony Brook University is one of the 62 research universities that comprise the Association of American Universities (AAU), an invitation-only organization of leading research universities in North America. There are a large number of biomedical researchers on campus. Many NIH-funded projects will benefit from having access to a cryo-EM facility. There will be two cryo-EM faculty members including the applicant who has been at the university since 2010, but there is no cryo-EM facility on campus. We therefore request a 200-KV cryo-TEM equipped with a direct detection camera. The new instrument is compatible with several existing automation software. Thus acquisition of the new microscope will enable cryo-EM research, increase productivity, and satisfy the need of this large life science community.

Title: Novel Statistical Methods in Analyzing Microbiome Data for Longitudinal Study Abstract: Recent research demonstrates that changes in the microbiome can have considerable health implications such as malnutrition, asthma, obesity, diabetes, and other conditions. This has further promoted substantial interest in the microbiome from both basic and clinical perspectives, and prospective longitudinal studies have been conducted to probe the mechanisms on how the microbiome affects health and disease. However, the special structure and characteristics of high-dimensional compositional microbiome data complicate effective analysis of microbiome data. In particular: 1) microbiome data is compositional; 2) microbiome data is high dimensional; 3) bacterial taxa are related evolutionarily by a phylogenetic tree; and 4) microbiome compositions are often quantified as sparse compositional data vectors (sparse vectors of proportions with unit sum). Limitations of proper statistical methods to analyze this unique high dimensional data hinder our ability to make inferences or draw conclusions about the role of the microbiome in human health and disease. Motivated by the challenges we have encountered during collaborative longitudinal microbiome studies on the effect of altered microbiome on the development of Type I Diabetes, we propose to accomplish the following specific aims: (1) to design a framework to compare the temporal changes of microbiome between groups; (2) to develop longitudinal mediation models to infer causality in microbe-induced complex trait/disease (diabetes) studies; (3) to develop a unified, powerful, and robust statistical framework to test the association between bacteria taxa and diseases/traits in microbiome studies; and 4) to develop, distribute and support the freely available software packages for the methods we develop. The methods will be evaluated through analytical approaches, computer simulations, and applications to multiple real datasets. The long-term goals of this application are to develop and implement novel statistical methods to study the dynamics of the microbiome composition, to identify key bacterial species that affect susceptibility to complex traits, to apply these methods to facilitate ongoing studies, and to disseminate these tools to the general research community.

? DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine is the host laboratory offering to provide a portion of the time on their JEM3200FSC and JEM2200FS electron microscopes for a cryo-electron microscopy (cryoEM) and tomography (cryoET) data acquisition facility Consortium. Both instruments are equipped with a field emission gun and an in-column energy filter, while the JEM2200FS also has a Zernike phase plate attachment. Both instruments are currently equipped with DE20 and DE12 cameras operated in integrating mode. These microscopes have been used productively for a variety of biological specimens, including macromolecules, molecular machines and cells, at state-of-the-art resolution. Our facilities will be allocated for he proposed Consortium while taking into account the ongoing research in the host institution. Our lead PI, Wah Chiu is a recognized expert in pushing cryoEM and cryoET beyond current boundaries and has decades of experience in leading several NIH funded research centers and academic training programs. There are 11 participating institutions across the USA, led by investigators who have a research track record in cryoEM and/or cryoET. Day-to-day operation will be carried out by a part-time cryoEM/ET scientist and IT staff. Both in person and remote data collection protocols will be used for distant users. The Consortium will provide travel funds for the distant users. We request funding for a Direct Detection Device (DDD) operated in counting mode to meet the needs of all types of specimens. The host Institution will provide an administrative assistant to support user visits and various Consortium activities, and matching funds to purchase the DDD and adequate hard drives for short-term data storage. The participating PIs will be involved in formulating the specific policies for Consortium governance and developing training activities. Scheduling will be allocated evenly in the first year among participants with some flexibility based on specimen readiness. Allocation of microscope times and priorities will be assigned in the second year and beyond based on recommendations of an external microscope allocation committee appointed by all the PIs to avoid conflict of interest. Al PIs will meet quarterly on WebEx to discuss technical and administrative issues, and we will hold an annual user meeting. An annual review of all aspects of the Consortium operation and the data outcomes of the users will be conducted by an advisory committee to assure the highest productivity and usage of the proposed facility. The membership of the participating institutions will be dynamically reviewed by all the PIs with the inputs from the external advisory committee after the second year of our operation.

? DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine is the host laboratory offering to provide a portion of the time on their JEM3200FSC and JEM2200FS electron microscopes for a cryo-electron microscopy (cryoEM) and tomography (cryoET) data acquisition facility Consortium. Both instruments are equipped with a field emission gun and an in-column energy filter, while the JEM2200FS also has a Zernike phase plate attachment. Both instruments are currently equipped with DE20 and DE12 cameras operated in integrating mode. These microscopes have been used productively for a variety of biological specimens, including macromolecules, molecular machines and cells, at state-of-the-art resolution. Our facilities will be allocated for he proposed Consortium while taking into account the ongoing research in the host institution. Our lead PI, Wah Chiu is a recognized expert in pushing cryoEM and cryoET beyond current boundaries and has decades of experience in leading several NIH funded research centers and academic training programs. There are 11 participating institutions across the USA, led by investigators who have a research track record in cryoEM and/or cryoET. Day-to-day operation will be carried out by a part-time cryoEM/ET scientist and IT staff. Both in person and remote data collection protocols will be used for distant users. The Consortium will provide travel funds for the distant users. We request funding for a Direct Detection Device (DDD) operated in counting mode to meet the needs of all types of specimens. The host Institution will provide an administrative assistant to support user visits and various Consortium activities, and matching funds to purchase the DDD and adequate hard drives for short-term data storage. The participating PIs will be involved in formulating the specific policies for Consortium governance and developing training activities. Scheduling will be allocated evenly in the first year among participants with some flexibility based on specimen readiness. Allocation of microscope times and priorities will be assigned in the second year and beyond based on recommendations of an external microscope allocation committee appointed by all the PIs to avoid conflict of interest. Al PIs will meet quarterly on WebEx to discuss technical and administrative issues, and we will hold an annual user meeting. An annual review of all aspects of the Consortium operation and the data outcomes of the users will be conducted by an advisory committee to assure the highest productivity and usage of the proposed facility. The membership of the participating institutions will be dynamically reviewed by all the PIs with the inputs from the external advisory committee after the second year of our operation.