CENTRALIZED RESEARCH INSTRUMENTATION

Biomedical research is a central part of the research activity in Puerto Rico. The Centralized Research Instrumentation (CRI) Cores impact the development of the INBRE network by supporting further integration with existing as well as newly created infrastructure at mentoring institutions and other affiliated institutions and programs. To fulfill the aims of this proposal, and to remain as a meaningful infrastructure support to the biomedical research enterprise in Puerto Rico, we will continue to maintain and reinforce the existing CRI Cores that : (1) support research applications in metabolomics, genomics, proteomics, and protein structure for the INBRE network and (2) promote usage of existing capabilities and specialized technologies to enable competitive studies by INBRE Investigators. The CRI Cores specific aims are as follows:

Specific Aim 1: Reinforce and enhance access to state-of-the-art core research instrumentation for nurturing new and expanding established research collaborations between the CRI investigators, students, and faculty from Puerto Rico, the Caribbean, and other IDeA states.

Specific Aim 2: Ensure the effective dissemination of CRI Cores technologies via theoretical and practical workshops, mentoring researchers in experimental design and best practices in the use of the resources that are available to them.

CRI Core Activities (2016-2021),

PR-INBRE Sequencing and Genotyping Facility (SGF):

Overview. Since August 2011, the SGF has been led successfully by Dr. Riccardo Pappa, Assistant Professor of Biology at the UPR Rio Piedras Campus. Dr. Pappa is proficient in the operation of the SGF instruments and NGS data analysis, since he uses these technologies in his own research project. The SGF was officially created in 2006 through a combination of funds from different grants, including NCRR AABRE Grant #P20 RR16470, NIH-SCORE Grant #S06GM08102, NSF-CREST Grant #0206200, and NINDS-SNRP U54 NS39405 and institutional matching funds. The main goal of the SGF is to provide researchers, students and faculty from Puerto Rico and the Caribbean access to state-of-the-art equipment, technology and technicians who are committed to helping investigators produce the highest quality data. Presently, the SGF is the largest and most active genomics facility in Puerto Rico. The Sequencing and Genotyping Facility (SGF), is designed to: 1) provide students and researchers in Puerto Rico with access to advanced equipment for collecting high-quality automated sequence and genotype data; 2) training and advising researchers and students island-wide on how to obtain, archive, and analyze data; and 3) create an alliance of researchers and students who share core research technology and who work together to strengthen scientific infrastructure and research competitiveness within Puerto Rico.

The SGF has kept abreast with the newest technology available, by acquiring an Illumina NextSeq 500 system with INBRE funds, providing these to its users. The SGF moved its operations to a larger and more modern laboratory space in the UPR Molecular Sciences Building located in Rio Piedras, PR in March 2014; thus enabling the SGF to fully accommodate the NGS and accessory instrumentation acquired over recent years. The SGF currently has over 30 main researchers and over 100 graduate and undergraduate students as users. The SFG will conform to the PR-INBRE charge back policies as defined in the CRI Business Plan, so as to enable the SFG to bill for services and prioritize requests for services through a computerized system, where each user will have a personalized account for service requests.

 

Table 1. Proposed Training Program for Sequencing and Genotyping Facility (SGF) Users

Type of User Training to be Offered Services to be Provided Other Requirements
Undergraduate (UG), Graduate (G) students, technicians, postdoctoral trainees and faculty from the UPR System or INBRE-affiliated institutions Sample preparation required for Illumina Next-Generation Sequencing (NGS), analysis of sequencing results. Includes, DNA isolation, quantitation, QC, gel electrophoresis, Bioanalyzer and other accessory equipment use. For G students, technicians, postdocs and faculty, same as above but with all hands-on accessory applications leading to QC and bioinformatics aspect of NGS. For UGs, five-person training in NGS techniques. Accessory equipment use is limited to trainer. For G students, technicians, postdocs and faculty, same as above but trainees will go through hands-on QC preparation and analysis. Users are required to go through SGF terms and necessary safety procedures before proceeding.

 Proposed Activities. Building on the growth experienced by the SGF in recent years, and the addition of Next Generation Sequencing (NGS) capabilities and outreach to network users, we are developing an undergraduate course schedule that will fully prepare laboratory workforce to effectively and efficiently use these cutting-edge technologies. In the proposed renewal period, the SGF will introduce hands-on training and technical aspects of NGS technology to the existing UPR Rio Piedras, Department of Biology Laboratory of Genetics (Biol 3350) course, currently offered to undergraduate biology students at the UPR Rio Piedras Campus. The SGF will continue to provide support in the preparation and submission of grant applications to the NIH, National Science Foundation (NSF), the National Aeronautics and Space Administration (NASA), the US Department of Agriculture (USDA), and other agencies to INBRE network users and will continue to provide support for the preparation of scientific, peer-reviewed manuscripts.

4b. INBRE/RCMI Genomics Translational Research Unit (GTRU)

Overview. The GTRU is led by Dr. Carmen L. Cadilla, Professor of Biochemistry at the UPR-MSC. Located presently in two sites: at the UPR School of Medicine and the Puerto Rico Cancer Center, the GTRU is supported an NIMHD grant (G12-MD 007600) to establish an RCMI Center for Genomics in Health Disparities and Rare Diseases (CGHDRD) and supplemental funding from the NIMHD PRCTRC grant. Dr. Cadilla has a broad background in Molecular Biology and Genetics with specific training and expertise in key aspects of analysis of different types of genomes. The GRTU supports projects that contribute to the understanding of genetic disorders affecting individuals of Puerto Rican ancestry, applying genomic technologies in the study of human diseases and/or use animal models of disease, or requiring applications in pharmaco-genomics as well as gene regulation mechanisms. The GTRU provides services and expertise to INBRE investigators in Next Generation sequencing technologies (with the Ion Proton and Ion Torrent instruments), Affymetrix microarray applications, real-time qPCR for expression analysis and Taqman Genotyping assays. The GTRU technician is Ms. Jessicca Renta, who is supported by RCMI funding. Ms. Renta has an MS in Microbiology and 19 years of experience in supporting research efforts in the areas of genetics and genomics. Ms. Renta’s contributions to users’ research projects have been recognized by co-authorship in 20 peer-reviewed publications.

The Metabolomics Research Sub-Core (MRC) was established as a part of the CRI in the fall of 2011. Dr. Nataliya Chorna developed non-targeted neurolipidomics analysis using a Gas Chromatographer/Mass Spectrometer (Agilent) instrument. These methodologies were integrated with the Human Genetics and Genomics Core and transferred to the UPR Medical Sciences Campus in the summer of 2015. This enabled the CRI to address functional genomics questions studied by investigators in PR-INBRE network institutions.   Dr. Chorna has cross-trained in genomic and/or proteomic methodologies so she can provide services in these areas as well. A charge-back system for services was implemented according to the CRI Business Plan as well as the prioritization of these requests.

Proposed Activities. The RCMI Human Genetics and Genomics Core laboratory strives to provide a multidisciplinary collaborative research program that supports local, national and international scientists working in pharmacogenomics, human genetics and genomics and molecular biology. This goal is met by providing efficient and specialized core laboratory services including technical support, consulting (with Dr. Cadilla serving as a mentor for some users) and training.

Resources Offered. Drs. Cadilla and Chorna, with Ms. Jessicca Renta, have complementing expertise that enables them to support research projects employing genomic and metabolomics technologies in the areas of emphasis in the PR-INBRE (i.e. Neuroscience, Molecular Medicine, Drug Development, Environmental and Behavioral Sciences). In addition, these core labs have established collaborations with institutions and research groups that can assist in the analysis of genomic data, such as the Pittsburgh Supercomputing Center (Professor Alex Ropelewski and Dr. Hugh Nicholas) and Dr. Ricardo González Méndez (UPR MSC Sch of Medicine). Analysis software, such as the Partek Genomics Suites and Ingenuity Pathway Analysis, is provided to users so they can perform basic analyses of array-based experiments as well as next generation sequence data. In order to better address core service needs in genomics, we will continue to provide the following services: DNA or RNA isolation; low temperature storage of DNA, RNA or protein samples, cells or tissues; quality control of RNA for expression analysis; mutation analysis by real-time PCR; semiconductor sequencing (genome, exome, transcriptome and amplicon (targeted) sequencing); expression analysis by Affymetrix GeneChips; assessments of metabolic changes at cellular and organism levels; and real time PCR. Support for training workshops in the technologies and related methodologies by the core will be continued. These will include theoretical and hands-on workshops, scientific seminars, lectures.

Table 2. Proposed Training Program for Genomics Translational Research Unit Core Laboratory Users

Type of User Training to be offered Services to be provided Other requirements
Undergraduate (UG), Graduate (G) students, technicians, postdoctoral trainees and Faculty users from the UPR System or INBRE Network institutions. DNA isolation and quantitation (manual), PCR, real-time PCR, gel electrophoresis, primer design, analysis of DNA sequencing results.

 

 

One-on-one or small group training in basic techniques. Microarray and Next Gen DNA Sequencing, the RCMI technician will run the samples and results will be provided to users. Training may be provided. Users will be required to have basic instruction in the responsible conduct of research in their relevant areas of research. Approval from relevant institutional (IRB, IACUC, etc.) regulatory committees required.
Faculty users from the UPR System or INBRE Network institutions. Introduction to metabolomics technology, extraction of fatty acids from blood, samples centrifugation, evaporation, trans-esterification, separation by GC/MS and bioinformatic analysis of obtained data. Small group (maximum of 10 trainees) will receive training on blood metabolomics. Faculty users from the UPR System or INBRE Network institutions.
Faculty users from the UPR System or INBRE Network institutions. Extraction of metabolites, as described above. Training will be provided to the investigators, their technicians, postdocs, and students. Faculty users from the UPR System or INBRE Network institutions.

Table 3. Major Equipment and Services Presently Provided through the PRCTRC/RCMI GTRU

Instrument Services Provided
+Ion Torrent Personal Genome Machine (PGM) and *Ion Proton Semiconductor Sequencers. Includes *accessory equipment for library preparation, QC and template preparation for these two instruments. Next Generation semiconductor sequencing chemistry, 30 Mb to 2Gb (PGM) total base reads in 200 or 400 base read modes; Ion Proton: 200-base fragment reads, 12 Gb total base reads. Applications supported include: RNA Seq (Transcriptome Sequencing), Amplicon Sequencing, whole genome sequencing (WGS), exome sequencing, ChIP-Seq
¨ABI 3500 and Prism 3130 Genetic Analyzers Sanger DNA Sequencing, Fragment analysis (microsatellite, etc)
¨Affymetrix Scanner 3000 7G System Affymetrix GeneChips – Expression and SNP Analysis, ChIP on chip
¨Biorad FX Molecular Imager Phosphor Imaging with the Molecular Imager/Scanner
¨Qiagen QIACube System Automated purification of RNA, genomic DNA, plasmid DNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup
Agilent Bioanalyzer Lab on a chip applications for RNA, DNA and protein capillary electrophoresis, QC steps in NGS or microarray applications
**ABI StepOne Plus, Real-Time PCR applications, Expression analysis, Genotyping, QC steps in NGS or microarray applications, etc
DQuant Studio 6 Real-Time PCR applications, Expression analysis, Genotyping, QC steps in NGS or microarray applications, etc
**epMotion 5070, Automated liquid handling systems designed to automate routine pipetting tasks to free up researcher’s time. It is one of the most accurate pipetting stations by virtue of automation and helps users eliminate manual pipetting errors and maximizes the reproducibility of assays.
**Beckman DU730 UV/Vis Spectrophotometer Spectrophotometric analysis and assays for up to 6 samples at a time, includes Peltier temperature control capabilities.
**Perkin-Elmer LS 55 Luminescence Spectrometer Fluorometric analysis and assays.
**Odyssey Imaging System/MousePod Infrared dye applications (Western blots, Mobility Shift Assays, stained tissues and animal imaging, etc.)

*Acquired through RCMI Grant 2U54MD007587; +Acquired with PR Science and Technology Matching funds to UPR-MSC.¨Acquired through NIH NCRR Grant G12RR003051; DAcquired through INBRE Grant P20 GM103475;**Acquired through NIGMS-RISE Administrative and ARRA Supplements.

INBRE ChEMTox In vitro Biotesting Facility:

Overview. The Universidad Metropolitana (UMET) ChEMTox In vitro Biotesting Facility is directed by Dr. Beatriz Zayas, Professor of Toxicology. Housed in the School of Environmental Affairs at UMET, the facility is located in Laboratory IC-125, Research Building, UMET. The laboratory provides services for the screening of biological activity of novel chemical compounds and nanoparticles with potential therapeutic applications. Its goal is to support scientists in their research of drug design and development as well as those interested in characterizing the mechanism of action of natural products or new materials. In addition to serving the scientific community, the facility is aligned with the academic and educational goals of the university, serving as a training site for undergraduate and graduate students in science.

Local scientists from different academic institutions in Puerto Rico, as well as scientists from the National Cancer Institute (NCI) in the US, have used these in-vitro testing services. The in-vitro analysis services focus on key cellular hallmark events that provide insights on the toxicity and general mechanism of action of the tested compounds. Analysis can be performed relatively quickly as compared to traditional methods. Results obtained from the testing facility have served to support the patent of novel compounds in 2012 (US patent No. US 8,124,770 B2) as well as in peer-reviewed publications with collaborators from the NCI and from universities in Puerto Rico.

Proposed Activities. Services currently offered at the ChEMTox include: HPLC/MS characterization of substances, determination of 50% Inhibition Concentration (IC50); apoptosis induction by Annexin V assay; cell cycle arrest assay; DNA Fragmentation assay; Mitochondrial permeability and activation of Caspases (3,7,8, &9). New toxicological assays are being incorporated. Services are offered, based on scientific collaborations or as a fee per service payment, giving the client the flexibility to choose assays that are relevant to their individual needs.

The In-vitro biotesting facility is in the process of expansion. The current 500 sq ft facility will be expanded to approximately 1500 sq ft laboratory. A variety of instrumentation is available including: High Performance Liquid Chromatography (HPLC), Mass Spectrometer, Fluorometer, Differential microscopy readers (allowing for measurement of a variety of cell response endpoints), and equipment and supplies required for cell culture.