Scientific Cores

Cell Separation and Culture (CSC) Core

The Cell Separation and Culture Core provides vital services to Center members.  The benefits of the Core are several-fold. First, it provides services that are frequently utilized by numerous funded investigators, thereby allowing sharing of resources and cost reduction. Second, it fosters an environment for collaboration and growth of investigators. Third, it provides a mechanism for development of new techniques that will benefit the investigators.

See Prioritization of use of the Core section for the criteria and rules applying to the use of the Core. For cell culture services, contact Dr. Xiu Yan Li(xiuyan@usc.edu) Research Associate of the Core. For use of Cell Separation resources, contact Dr. Zhang-Xu Liu (zxliu@usc.edu, co-Director of the Core.

Goals
The long-term objectives of the Cell Separation and Culture Core are to:

  1. Provide high quality, consistent services needed by a large number of independently funded investigators to improve research efficiency by sharing resources and lowering costs.
  2. Foster an environment for interdisciplinary collaboration and growth.
  3. Train investigators at USC and elsewhere in techniques for cell isolation and separation.
  4. Develop new services that the funded investigators and junior faculty will likely use in the future.

Facilities and Resources

Space:
The Cell Separation and Culture Core is located on two floors of the Hoffman Memorial Research Building. The Culture component occupies 400 sq ft in two rooms on the fourth floor of the Hoffman Memorial Research (HMR) building that also houses the research laboratories of many of the Center members. One 265 sq ft room (HMR-412) contains two hoods, four incubators, one autoclave, two liquid nitrogen tanks, one water distiller, and one tissue culture microscope. This room serves as the common workroom for investigators that utilize the Core. A separate 133 sq ft room (HMR-410A) used for sterile surgical procedures also contains one hood, one refrigerator/freezer, and one refrigerated table-top centrifuge (Beckman CS15R). The Separation component of the Core occupies 600 sq ft in a room (HMR-803) adjacent to Dr. Zhang-Xu Liu’s laboratory.

Equipment:
The following equipment and resources are currently available to the Cell Separation and Culture Core:

  1. Incubators (Forma Scientific, models 3110, 3326, Symphony) – four, 2 for primary cultures, 2 for cell lines
  2. Biosafety Cabinets (Labconco, Baker) – four
  3. Refrigerator/freezer – one
  4. Microscope – two, one is a light microscope for cell counting and viability check by hemocytometer (Motic), one is capable of photomicroscopy for close examination of cells (Nikon Diaphot).
  5. Vacuum pumps (Air Cadet) – three
  6. Water bath (Baxter) – two
  7. Centrifuge (Beckman GS15R, Labnet Z400K) – one
  8. Liquid nitrogen storage – two
  9. Water distiller (provided by Dr. Stiles’ lab) – one
  10. Autoclave (provided by Dr. Stiles’ lab) – one
  11. BD FACSCalibur flow cytometer – one
  12. Miltenyi Biotec AutoMACS Pro Separator – one
  13. BD FACSVerse Flow Cytometer – one

Functions and Activities

Overview:
The combined Cell Separation and Culture Core provides two major types of services: 1) cell separation to allow functional analysis of different cell types; and 2) isolation and culture of hepatocytes from normal and diseased rodents and cell line banking.  In addition, we plan to develop the following services this year: 1) establish stable hepatocyte cell lines that express the humanized Cas 9 to facilitate gene knockout studies using the CRISPR system; 2) establish service for MACS isolation of liver progenitor cells using cell surface markers CD133, CD49f and CD24; 3) establish protocols for culturing immortalized pluripotent stem (iPS) cells.

Dr. Bangyan Stiles serves as Director of the Core, replacing Shelly Lu in August 2014, and Dr. Zhang-Xu Liu serves as Associate Director. The Core will continue to maintain one 12-month technical support shared between two individuals.  One individual is primarily responsible for the established service (80%) and the other individual (20%) is responsible for back-up and for the research and development. The FACS and MACS facilities are self-serve facilities and Dr. Liu provides technical training for new users.

Services:

Cell Culture component of the Core:
The Cell Culture component of the Core provides fee for services for the USC Research Center for Liver Disease.

Existing Services:
The Core provides isolation and culturing of rat and mouse hepatocytes from normal and diseased livers, and cell line banking. The ability to isolate and culture hepatocytes of consistently excellent quality has been invaluable to many Liver Center members so that they don’t need to have this technique established in their own labs. The following are the services offered by the Core:

  • Isolation and culture of primary rat hepatocytes from normal and disease models (such as bile duct ligation and alcohol fed models). Isolation of primary rat hepatocytes is done aseptically according to the method of Moldeus et al (Moldeus P, Hogberg J and Orrenius S. Isolation and use of liver cells. Methods Enzymol. 51:60-70, 1978). The method is based on collagenase digestion and separation of viable liver parenchymal cells, which can be further improved with Percoll. The cells are cultured in DME/F12 medium containing high glucose (3151mg/L), 1mM methionine, 10% FBS, hydrocortisone (50nM) and insulin (1µg/ml) on dishes precoated with rat tail collagen, unless otherwise requested (e.g. matrigel precoated plates or collagen overlay), and incubated at 37˚C in 5% CO2, 95% air. This protocol is very well established  and has been in use in the core for the last several funding cycles.
  • Isolation and culture of primary mouse hepatocytes. Cultured or freshly isolated mouse hepatocytes are prepared from C57B (both 6J or N strains whichever is needed), Swiss/Webster, Balb/c or C3H/HE mice. Other strains can also be used but will need to be provided by the investigator. Transgenic and knockout mice are also provided by individual investigators. Murine hepatocytes are prepared as described (DeLeve, L.D.  Hepatology 24:830-837, 1996).
    • Isolate and culture of hepatocytes from various transgenic and knockout mice as well as mice treated with toxicants or subjected to bile duct ligation. In many instances the procedure must be modified in order to maximize the yield of hepatocytes. The amount of collagenase used, duration of perfusion and flow rate need to be adjusted for each case. This is a routine service provided by the Core.
  • Maintenance and availability of cell lines. The following provides a brief description of each:
    293 – primary human embryonal kidney transformed by sheared human adenovirus type 5 DNA.
    AML12 – an immortalized, non-transformed murine hepatocyte cell line suitable for transfection studies.
    CaCo-2 – a human colon carcinoma cell line, can be grown as polarized cell and may be useful for studies of transport in intestinal cells.
    H4IIE – a rat hepatoma cell line.
    HeLa – a cervical carcinoma cell line often used for gene expression studies.
    HepG2 – a well-differentiated human hepatoblastoma cell line, frequently used as a model for the study of human hepatocytes.
    HT-29 – a human colon carcinoma cell line with a mutant p53 antigen
    HuH-7 – a well-differentiated hepatoma cell line often used in hepatitis virus research.
    NIH3T3 – a fibroblast cell line commonly used for oncogene transformation assays.
    RAW 264.7 – a mouse monocyte/macrophage cell line
    RKO – a human poorly differentiated colon carcinoma cell line with wild type p53

    • All of these cell lines (except Huh7) are available through ATCC. The Core has also stored specialized cell lines for Center investigators, such as CYP2E1 overexpressing HepG2 cells and an accompanying vector control expressing HepG2 cells, and CD49f+ liver progenitor cells from MAT1A knockout mice.
  • Distribution of human hepatocytes.  The Core is also in charge of distributing human hepatocytes whenever they are available. CellzDirect provides these specimens free of charge to Center investigators.
  • Non-parenchymal cells isolation. The Non-Parenchymal Cell Core is a component of the Southern California Alcoholic Liver and Pancreatic Disease Center (ALPD). This core provides Kupffer cells, stellate cells, and sinusoidal endothelial cells from normal and diseased rat and mouse models.
  • Training.  The Core provides training to investigators at USC or outside of USC that are NIH-funded.

Monitoring of Quality control:
The quality control for primary cultures of hepatocytes are:

  1. Measure viability by trypan blue exclusion at the end of the isolation procedure and only those >85% are provided to users.
  2. The Core routinely keeps two 60x15mm plates of the cultured hepatocytes to assess cell viability the next day by LDH release and for contamination check. Our LDH release is <10% for preparations that had >85% viability.

The quality control for cell lines are:

  1. Mycoplasma testing (done at Norris Cancer Center Core facility) every three months. Cell lines tested strongly positive are discarded and replaced from ATCC. Cell lines tested weakly positive are treated with mycoplasma removal agent and retested. Thus far, contamination has occurred only once and the cell lines were replaced.
  2. Minimizing passage effect. Passage number affects different cell lines differently. Cells can undergo genetic drift at high passage. To avoid this we record passage number of all cell lines and bank early passage cells. We also routinely check morphology of the cells and their growth rates (doubling time) and once a change is noted, the cells are discarded and an early passage vial is thawed for replacement. When all early passage vials are exhausted, we replace the cell line by obtaining fresh vials from ATCC.
  3. For cell lines that have been genetically manipulated, the members are encouraged to perform routine karyotypings.  For cell lines that are banked with the center, we will perform karyotyping on an annual basis.

Research and Development (New services to be implemented – planned to begin in late 2015):
Looking toward the future, discussions between Dr. Stiles and the Center leadership and Executive Committee lead to the selection of the three new services for development by the Core based upon their intimate knowledge of the directions of the research of members in the four Themes of the Center.

  1. Establishing HepG2 and Huh7 cell lines stably expressing CAS 9.  Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas) are essential components of nucleic acid-based adaptive immune systems that are widespread in bacteria and archaea.  In recent years, these components have been developed as valuable biotechnology tools for introducing gene deletions/mutations in somatic cells. To utilize this system, introduction of the cas nuclease capable of cleaving double stranded DNA and the guiding nucleotide sequences that guide the nuclease to specific DNA locus is necessary.  Recently, Dr. Stiles’ lab successfully introduced the humanized Cas9 nuclease gene to the Pten null liver progenitor cells (Rountree,..Stiles Stem Cell 2009 and data not shown) and established cell lines that stably express the Cas 9 nucleases for gene targeting.  Such cell lines allow us to introduce designed guiding nucleotide sequences to target any gene sequence to delete a portion of the gene sequence of that particular gene.  We plan to introduce the Cas 9 gene to Huh 7 and HepG2 cells, two human hepatoma/hepatoblastoma cell lines we already have in our storage.  These cell lines will be made available to the center members to perform their gene deletion studies.
  2. Establishing service to isolate liver progenitor cells using MACS based cell surface marker sorting.  In addition to the existing service, the core will establish service to isolate murine liver progenitor cells from injured and diseased livers in the next grant cycle. Dr. Stiles is experienced in the isolation of liver progenitor cells using her liver Pten deletion model (Rountree et al Stem Cell 2009). The Core facility is equipped with the equipment (MACS) to isolate liver progenitor cells using cell surface markers. We plan to develop this service for the Center members.  First, we will develop sorting ability for the CD49f+CD45- cell populations.  Then we will add the ability to sort for additional surface markers (e.g. CD133 and possibly CD24). For service, two operation models will be considered.  A full service will cover the entire procedure from digestion of the liver to cell isolation.  In this case, the investigators will provide the livers, Dr. He will perform the procedure and return the CD49f+CD45- cells.   Partial service model may also be considered where Dr. He or Li perform liver cell isolation and the investigators using the core MACS will sort and isolate the CD49f+CD45- cells by themselves.  Training will be provided.  Chargebacks will be established to cover supplies.
  3. Establishing protocols for culturing iPS.  To anticipate future development, we plan to develop protocols to culture immortalized pluripotent stem cells (iPS). The ability to differentiate embryonic stem cell (ES) or iPS to hepatocytes allows researchers to understand the link between genetics and liver function in cellular systems derived from patients. USC Institute of Stem Cells and Regenerative Medicine provides the service to derive iPS for investigators but not continued culturing after the derivation.  Therefore, we will develop technical expertise to maintain these iPS cultures for Center members who derive them in the Stem Cell core.  Dr. Stiles has experience working with ES cells (Stiles et al MCB 2002) and the center technician will receive training to establish this service.  Charges for these new services will be established once available.

Cell Separation component of the Core:
The Cell Separation component of the Core provides assistance to Center members in the use of Core’s following equipment to isolate cell subtypes and analyze their function. There is currently no fee to use these instruments and Dr. Liu is in charge of training users and providing assistance when needed.

  • Miltenyi Biotec AutoMACS Pro Separator.  The AutoMACS Pro Separator (MACS) is a benchtop automated magnetic cell sorter that can be used for the isolation and purification of a wide variety of cell subtypes from blood, bone marrow and liver non-parenchymal cells. The MACS’ automated system can handle up to 6 samples simultaneously, under standardized conditions, and with sensor technology that allows monitoring of the instrument’s status. The instrument is operated via touch screen with intuitive user menus, which will enable rapid training of all users, and has 10 preset programs. The pre-set programs can vary flow rates across the magnets and direct flow over one or two columns, so that populations of cells can be positively or negatively selected (e.g. NK1.1 positive versus NK1.1 negative non-NK cells) and can also be isolated with high and low expression of antigens (e.g. sinusoidal endothelial cells from different parts of the liver lobule). For the renewal cycle, we will also use this sorter to establish and provide the liver progenitor cell sorting service to negatively select for CD45- and positively select for CD49f (or other) markers. The ability to handle up to 6 samples simultaneously allows isolation of a cell type from multiple sites from a single animal (e.g. blood, bone marrow and liver homogenate) or from multiple animals. The new services will be supported by Dr. He once established.
  • FACSCalibur flow cytometer.  The BD FACSCalibur flow cytometer offers a unique modular approach to flow cytometry and allows users to perform cell analysis in an innovative single automated bench-top system. The BD FACSCalibur system is compact and simple to operate, which enable fast, easy, and accurate results for routine applications in up to 4 color analysis of cell subtypes, based on the fluorescence tagged antibodies against cell surface markers in the field of molecular biology and immunology. The BD FACSCalibur system is complemented by a broad suite of intuitive software solutions to streamline analysis for a wide range of applications, including:
    • Detection of cell surface antigen expression and enumeration of lymphocyte subsets
    • Immune function studies (detection of various intracellular cytokines and  secondary mediators)
    • Detection of the mode of cell death (necrosis or apoptosis)
    • Cell cycle analysis
    • Examine transgenic products in both in vitro and in vivo, particularly GFP or related fluorescent cell surface antigens
    • Characterization of stem cells
  • FACSVerse flow cytometer. During the past funding year, A 3 laser 8 color BD FACSVerse Flow Cytometer was purchased, which offers remarkable performance for over 4 color research application.  The BD FACSVerse system supports simultaneous detections of up to 8 different fluorescence-tagged antibodies (markers), and is complemented by newly designed intuitive FACSuite software solutions to streamline analysis for a wide range of advanced and complicated applications, including:
    • Detailed immunophenotying of human and mouse immune cells and subsets, usually requiring more than 4 colors for one sample analysis, particularly useful for limited amounts of purified cells (blood, tissues) from patients.
    • Functional studies of immune cells: intracellular cytokine detection for profiling of T lymphocytes (Th1, Th2, Th17), monocytes/macrophages, NK/NKT cells.
    • Detection of cell apoptosis and necrosis of specific cell types by multi-color parameters (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity).
    • Particularly useful in identification and characterization of stem cells/progenitor cells by stratified gating strategy under multi-color cell surface parameters.
    • Measurement of cytokines, chemokines, growth factors, and other key markers by BD Cytometric Bead Array (CBA)-based multiplex immunoassays to monitor the immune response in serum/plasma and culture media. The BD CBA system uses the broad dynamic range of fluorescence detection offered by flow cytometry. Each antibody-coated bead in the array has a unique fluorescence intensity.  These beads can be mixed and run simultaneously in a single tube, which allows users to quantify multiple proteins simultaneously. In comparison with traditional ELISA, such FACS based CBA method significantly reduces sample requirements and time. The BD CBA multiplexed assay allows detection of up to 30 proteins with sensitivity as low as 0.274 pg/mL using just 25 to 50 μL samples. This is particularly powerful for analysis of limited amounts of human samples of plasma/serum, immune cells and tissues in clinical research projects. Dr. Liu is using this approach to profile a panel of the inflammatory cytokines/chemokines and soluble cell surface markers in the plasma samples and cultured PBMCs from alcoholic hepatitis patients.  The BD FACSVerse flow cytometer is also equipped with BD FACSuite software.  The automated features of this software allows for faster and more accurate data analysis. In addition, keywords entered into BD FACSuite software are recognized in FCAP Array software during analysis, resulting in a simple, robust workflow for protein quantitation. This automated workflow also reads the Sample ID and Experiment Name directly from the data file, providing consistent identification of a sample throughout the entire workflow and verification that all data files are from a related experiment.
    • Supplemented with the existing 4 color basic model BD FACSCalibur system, the FACSVerse system will support users with much more detailed and advanced analysis of cell surface markers and intracellular mediators in our advanced immunology and molecular biology studies.

Personnel
Bangyan Stiles, Ph.D.   Director
Dr. Stiles (bstiles@pharmacy.usc.edu) took over as Director of Cell Separation and Culture Core, replacing Dr. Shelly Lu in August 2014. As the Core Director, she supervises the work of the Core’s Research Associates, oversees the utilization of the Core and chargebacks. She also oversees the development of new services. She reviews requests, judges priority and eligibility for the use of the Core and oversees allocation of funds and chargebacks, subject to approval and review by the RCLD Director and Executive Committee.

Dr. Zhang-Xu Liu, M.D., Ph.D.  Co-Director
Dr Liu (zxliu@usc.edu), Co-Director, is in charge of the Cell Separation component since its establishment and also has the responsibility for overseeing activities of the entire Cell Separation and Culture Core in Dr. Stiles’ absence. His work involves extensive use of the flow cytometric techniques. Dr. Liu is highly trained in immunology and is an expert in the flow cytometric analyses of isolated immune cells, and has extensive experience in cell cultures.

Xiu-Yan Li, M.D.  Research Associate
Dr. Li(xiuyan@usc.edu) has been the Research Associate of the Cell Culture Core since its inception in April 1995. She has over 30 years experience in tissue culture. She consistently obtains primary hepatocytes of the highest quality. She maintains rigorous quality control and contamination check for bacteria and mycoplasma are done on a regular basis. She is also responsible for keeping track of supplies, billing and generating monthly statements with the supervision of the Director of the Core.

Lina He, Ph.D.  Research Associate
Dr. He is a part-time (at 20% effort) Research Associate for the Core and covers for Dr. Li in her absence. She has more than 20 years experience in tissue culture work and was trained by Dr. Li in hepatocyte isolation. Dr. He will also be responsible for developing new techniques including the new cell lines expressing Cas 9, MACS isolation of liver progenitors and iPS culturing.

Management

Organization:
A brief description of available and new services provided by the Cell Separation and Culture Core is periodically distributed to all participants of RCLD via e-mail and is posted on the Center’s website.  All investigators are required to provide proof that they have the appropriate approval (e.g. IACUC and IRB approval) for the work requested. The following present the procedures:

  1. Preparation of primary cultures and freshly isolated rat and mouse hepatocytes from normal and diseased models:  Isolation of hepatocytes is done on an as-needed basis. The Core needs to be notified at least 24 hours ahead of time so that effort is made to have the cell preparation shared by as many investigators as possible. There is a limit on the number of investigators one cell preparation can serve – up to three if plating is required, but no limit if plating is not required as long as there are enough cells. Cells are attached ~2hrs after plating, which is the time that they are picked up from the Core (unless prior arrangements are made). Medium changes are done by individual labs. In case of disease models, the investigators provide the Core with animals and preparation of the hepatocytes is done by the Core.
  2. Cell line banking service:  Cell lines are thawed and plated as per request and the Core also freezes and stores special cell lines for various investigators. Protocols for caring of the cells are provided, mostly according to ATCC. PIs can order cell lines from the Core anytime and they are notified when cell lines are ready for pick up.
  3. Distribution of human hepatocytes:  We have an arrangement with CellzDirect to have them prepare and provide human hepatocytes to Center investigators free of charge (except for cost of shipping). To facilitate this service, CellzDirect notifies Dr. Stiles one day in advance when there are human hepatocytes available. Dr. Stiles in turn communicates with Liver Center investigators that have previously indicated an interest in using human hepatocytes. Cells are then distributed to these investigators. Human hepatocytes are typically delivered either in suspension in cold preservation medium or plated on either collagen gel or matrigel. The specific requirement of the substratum can also be communicated with CellzDirect. Typically, human hepatocytes in suspension are delivered to USC about 24 to 36 hours after livers are harvested. CellzDirect was purchased by Life Technologies in May 2010 and this arrangement has continued.
  4. Isolation of liver progenitor cells:  Isolation of liver progenitors will be done on an as-needed basis, following the same procedure as the ones already established for hepatocytes. At least 24 hours notification will be required for service to be rendered. Culturing of the isolated progenitors will be done by individual investigators, not part of the core service.

Prioritization of Use of the Core:

Priority will be given to NIH funded investigators, first Center members and then other USC investigators on a first come first served basis. Qualified users are investigators with peer-reviewed funding, as well as faculty with Center P/F Project or other start-up funds, plus individuals with non-funded but approved protocols by the research committee at USC. Investigators outside USC but funded by NIH can also use the Core; however, this is at a priority below USC-based investigators. Prospective users need to apply to the Executive Committee of RCLD for membership, which in case of approval qualifies them as a priority user, or determines their non-member user status. Dr. Stiles is in charge handling day-to-day prioritization of training and usage of cell culture services and Dr. Liu for the use of cell separation resources.

Costs:
There are charges for most of the services provided by the Culture component of the Core (see Table 1) to cover the cost of animals and basic supplies. For Center members the fee includes reimbursement of supplies and animal cost. For non-Center members, the fee also includes labor. When a cell preparation is shared by multiple investigators, the cost to the investigator is adjusted accordingly. Nearly all of the regular users of the services of this core are RCLD members with occasional use by others at USC or elsewhere. Currently there is no charge for use of the Cell Separation equipment. However, this may change depending on future budgetary constraints so that chargeback may be necessary to partially cover service contracts of equipment.  In addition, once progenitor cell isolation is available, we will develop a chargeback for supplies.

Table 1. Summary of Charges*
Table of Charges

Quality Management Plan:
The quality of the hepatocytes provided by the Core has received compliments from Center members and there have been no complaints of other services provided.  To formalize a quality management plan, we will conduct surveys on a yearly basis for frequent users.  For sporadic users, this survey will be handed out at the end of the service rendered.  The survey will include four parts: 1) quality of the product; 2) quality of the service; 3) handling of billing; 4) other communications.  This survey will be included in the annual review process with the Advisory Board members.

  • Quality of the product:  This survey will assess whether the quality and quantity of the product provided meet the requirement of the individual researchers.  This will include whether their needs for cells are met by the product provided.  Simultaneously, investigators will be reminded that users are required to cite the Core in their publications and such acknowledgements will be monitored.
  • Quality of the service: This survey will assess whether the quality of the service provided meets the requirement of the individual researchers.  This will include whether the time sensitive service requests are met, and whether the service is provided in a collegial manner.
  • Quality of the billing service:  Since the majority of core services will have charge backs, billing and record keeping is necessary.  This survey will assess whether the billing process is handled in a smooth and transparent manner.
  • Other Communications: Individual investigators are encouraged to provide feedback when deemed necessary.  In addition, we will request suggestions for developing new services.  For example, we plan to survey the need for alternative hepatocyte cultures, such as matri-gel or sandwich culturing.  If the need arises, we plan to develop these services in the future.

Benefits

The benefits of the Cell Separation and Culture Core are many-fold. First, the Core provides services, which are frequently utilized by numerous funded investigators, thereby allowing sharing of resources and cost reduction. Second, it fosters an environment for collaboration and growth of investigators. Third, it provides a mechanism for development of new techniques that will benefit the investigators.

Recent Publications
The following studies have addressed multiple and different areas of liver pathobiology and illustrate the vital role of Cell Separation and Culture Core in facilitating these research activities (Center members are in bold):

  • Yang HP, Ko K, Xia M, Li TWH, Oh P, Li J, and Lu SC. Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of GSH synthetic enzymes and contributes to cholestatic liver injury in mice.Hepatology 2010, 51:1291-1301. PMCID: PMC2908963. The Core isolated hepatocytes from mice that had undergone bile duct ligation (BDL). The BDL livers were fibrotic so that technique for isolating hepatocytes had to be modified in order to obtain sufficient yield. Without the expertise of the Culture Core research associate, this would have been difficult for individual labs.
  • Tomasi ML, Ramani K, Lopitz-Otsoa F, Rodríguez MS, Li TWH, Ko K, Yang HP, Bardag-Gorce F, Iglesias-Ara A, Feo F, Pascale MR, Mato JM, and Lu SC. S-adenosylemthionine regulates dual-specificity MAPK phosphatase expression in mouse and human hepatocytes. Hepatology 2010, 51:2152-2161. PMCID: PMC2905543. The Core provided mouse and human hepatocytes. This has greatly facilitated Dr. Tomasi’s research productivity, allowing her to successfully obtain her F32 after obtaining her Ph.D. degree and recently, her K01.
  • Ko K, Iglesias-Ara A, French BA, French SW, Ramani K, Tomasi ML, Lozano JJ, Oh P, He L, Stiles BL, Li TWH, Yang HP, Martínez-Chantar ML, Mato JM, and Lu SC. Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis and hepatocellular carcinoma in mice. Hepatology 2010, 52:2096-2108. PMCID: PMC3005187. The Core provided cell lines and isolated hepatocytes from Prohibitin 1 knockout mice. This paper was instrumental in securing a tenure-track position for the post-doctoral fellow Dr. Ko.
  • Than TA, Lou H, Ji C, Win S, Kaplowitz N: Role of cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) in the initiation of mitochondrial biogenesis and stress response in liver cells. J Biol Chem 2011 Jun 24; 286(25): 22047-22054. PMID: 21536665. PMCID: PMC3121349. The Core provided primary mouse hepatocytes to study the mechanism of acetaminophen-induced hepatotoxicity.
  • Tian Y, Sir D, Kuo CF, Ann DK, and JH Ou. Autophagy required for hepatitis B virus replication in transgenic mice. J Virol. 2011 Dec; 85(24): 13453-56. PMID: 21957292. PMCID: PMC3233133. The Core isolated primary hepatocytes from the transgenic mice for this study.
  • Xu J, Lai KK, Verlinsky A, Lugea A, French SW, Cooper MP, Ji C and H Tsukamoto. Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK. J Hepatol. 2011 Sep; 55(3): 673-682. PMID: 21256905. PMCID: PMC3094601. The Core prepared primary hepatocytes from the alcohol and HFD fed mice for this study.
  • Zeng, N., Xu, X., He, L., Galacia, V., Deng, C.-X and Stiles, BL (2011) Hyper-phosphorylation of eIF2α induced by PTEN deficiency promotes survival of hepatocytes under oxidative stress. Molecular Cancer Research  9(12):1708-17. PMID: 22009178. The Core isolated hepatocytes from Pten knockout mice.
  • Kao E, Shinohara M, Feng M, Lau MY and C Ji. Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes. Hepatology. 2012 Aug; 56(2):594-604. PMID: 22407670. PMCID: PMC3406240. The Core isolated primary hepatocytes and provided human hepatocytes for this study.
  • Shaker A, Binkley J, Darwech I, Swietlicki E, McDonald K, Newberry R and DC Rubin. Stromal cells participate in the murine esophageal mucosal injury response. Am J Physiol. 2013 Apr 1; 304(7): G662-72. PMCID: PMC3625876.  The FACS Sub-core is used to isolate stromal cells for characterization.
  • Wang L, Wang X, Xie G, Wang L, Hill CK and LD DeLeve. Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats. Journal of Clinical Investigation. 2012 Apr 2; 122(4): 1567-1573. PMID:22406533. PMCID: PMC3314456.  The FACS Sub-core is used to isolate sinusoidal endothelial progenitor cells for characterization.
  • Zhong Q, Shi G, Zhang Q, Zhang Y, Levy D and S Zhong.  Role of phosphorylated histone H3 serine 10 in DEN-induced deregulation of Pol III genes and cell proliferation and transformation. Carcinogenesis. 2013 Nov; 34(11): 2460-9. doi: 10.1093/carcin/bgt219. Epub 2013 Jun 17. PMID: 23774401. PMCID: PMC3888355.  The Core provided cell lines and isolated primary hepatocytes from the DEN-treated animal models.
  • Peng H, Dara L, Li TW, Zheng Y, Yang HP, Tomasi ML, Tomasi I, Giordano P, Mato  JM and SC Lu. MAT2B-GIT1 interplay activates MEK1/ERK 1 and 2 to induce growth in human liver and colon cancer. Hepatology. 2013 Jun; 57(6):2299-313. PMID: 23325601. PMCID: PMC3642222. The Core provided cell lines and isolated hepatocytes from MAT2B  knockout mice.
  • He S, Ni D, Ma B, Lee JH, Zhang T, Liang C et al.  PtdIns(3)P-bound UVRAG coordinates Golgi-ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex.  Nat Cell Biol. 2013 Oct; 15(10): 1206-19. doi: 10.1038/ncb2848. Epub 2013 Sep 22.  PMID: 24056303. PMCID: PMC3805255. The Core provided cell lines.
  • Li, C., Li, Y., He, L., Agarwal, A., Zeng, N., Cadenas, E., and Stiles, BL (2013) AKT regulates mitochondrial respiration by direct translocation .  Free Radical Biology and Medicine 60: 29-40. PMCID: PMC365403 The Core isolated hepatocytes from Pten knockout mice.  The Core provided isolated hepatocytes from Pten knockout mice.
  • Li, Y., Zeng, N., Galicia, V., He, L., Li, C., Han, D., Cadenas, E., and Stiles, BL (2013) Regulation of mitochondrial biogenesis by PI3K/AKT mediated ERRa induction. J. Biol. Chem. 288(30): 25007-24. PMCID: PMC3757167. The Core isolated hepatocytes from Pten knockout mice.
  • Yang HP, Cho ME, Li TWH, Peng H, Ko KS, Mato JM, and Lu SC. MiRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma. J. Clin. Invest. 2013, 123:285-298. PMCID:PMC3533284. The Core provided several cell lines used in this study and banked several cell lines created for this work.
  • Pastuszka MK, Wang X, Lock LL, Janib SM, Cui H, DeLeve LD and JA MacKay.  An amphipathic alpha-helical peptide from apolipoprotein A1 stabilizes protein polymer vesicles.  J Control Release. 2014 Oct 10; 191:15-23. doi: 10.1016/j.jconrel.2014.07.003. Epub 2014 Jul 10.  PMID:  25016969.  The Core provided cell lines used in this study.
  • Pirooz SD, He S, Zhang T, Zhang X, Zhao Z, Oh S, O’Connell D, Khalilzadeh P, Amini-Bavil-Olyaee S, Farzan M and C Liang.  UVRAG is required for virus entry through combinatorial interaction with the class C-Vps complex and SNAREs.  Proc Natl Acad Sci U S A. 2014 Feb 18; 111(7): 2716-21. doi: 10.1073/pnas.1320629111. Epub 2014 Feb 3.  PMID: 24550300. PMCID: PMC3932887. The Core provided cell lines used in this study.
  • Palian BM, Rohira AD, Johnson SAS, He L, Zeng N, Dubeau L, Stiles BL, Johnson DL.  Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.  PLOS Genetics 2014 in press.  The Core provided cell lines used in this study and isolated primary hepatocytes from AKT and Pten deleted mice.

Cell and Tissue Imaging (CTI) Core

The Core offers assisted imaging services, using confocal and fluorescence microscopy primarily with its Zeiss LSM 510 and Nikon TE-300 Diaphot systems as needed by Center members. Besides, Dr. Hamm-Alvarez’s Zeiss LSM 510 Meta NLO confocal and multiphoton system is available as an ancillary system for more advanced applications. In addition, a stand-alone workstation supports the imaging software of the Core’s two systems as well as other general-purpose packages. It also provides users on line access to the Core’s two systems for downloading and processing of their image files, while the microscopes are occupied by others users, thus alleviating bottlenecks forming on the Core’s microscopes by users with extensive post-acquisition image processing needs.

See Prioritization of use of the Core section for the criteria and rules applying to the use of the Core. For sign up and access to the Core contact Ms. MacVeigh-Aloni (macveigh@usc.edu), Research Specialist of the Core.

Goals
The long-term objectives of the CTI Core are to:

  1. Maintain the confocal and fluorescence microscopy facilities of RCLD.  We will maintain Core facilities according to rigorous standards of microscopy usage and image processing, using all relevant standards of quality control.
  2. Provide assisted confocal and fluorescence microscopy and image processing to Center members.  We will assist Center members to maximally utilize the Cores’ own facilities in the most efficient manner that enables them to achieve their research and experimental goals. We will also aid them in state-of-the-art image analysis and preparation of publication quality images.
  3. Provide education and training in diverse microscopy applications supported by the Core.  We will provide theoretical and practical seminars and workshops focused in areas of microscopy supported by the Core, including different modes of confocal fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET), plus multiphoton fluorescence microscopy and 3D-SIM.  We will also provide smaller customized workshops and hands-on-training for a limited number of motivated users who want to use the systems without assistance.  The Core Director will also assist members through individual consultations regarding the alignment of particular imaging applications to address specific research questions.
  4. Provide access and financial support for RCLD Center members to access USC Core imaging facilities enabling 3D-SIM, spinning disk confocal microscopy and multiphoton microscopy, beginning in December 2015 in the next funding cycle of RCLD.  While these instruments are available to all USC faculty on a fee-for-service basis in the respective University Cores, the School of Medicine has generously offered $50,000 annual subsidy, to be allocated to Center members to enable their access to these facilities. Thus, funds for access to these instruments will be disbursed through quarterly submission of a brief pilot grant application for a maximum of $2,500 (renewal upon resubmission of a progress report) to the CTI internal advisory committee (CTI IAC) comprised jointly of senior liver researchers and imaging specialists with expertise in the respective instruments.

Facilities and Resources

Space:
The Cell and Tissue Imaging Core occupies two adjacent 100 sq. ft. internal rooms located within a laboratory (HMR 610) on the 6th floor of the Hoffman Memorial Research Building (HMR), where a high concentration of Liver Center investigators’ labs are located. One room (HMR-610C) houses the Zeiss LSM 510 confocal system and the other (HMR-610B) the Nikon TE-300 Diaphot fluorescence imaging system. The rooms are in the same laboratory that also houses the Liver Histology Core (HMR-610A), which results in extensive interaction between users and staff of the two Cores.

Equipment:
Instrumentation currently available and offered to Center members is divided into two sections:  I. Dedicated Core Instrumentation and II. Instrumentation to be available through facilitated/subsidized access to Center members upon renewal of RCLD grant:

I.  Dedicated Core Instrumentation
Zeiss LSM 510 confocal microscopy imaging system: This system, acquired through an NIH shared instrumentation S10 grant (to Ookhtens and Hamm-Alvarez in 2007), consists of an AXIO Observer.Z1 motorized base microscope with a motorized scanning stage, attached to a laser scanning module (triple-VIS/405 UV), and it has capabilities for 4-fluorophore detection ±DIC or phase overlay, time-lapse and multi-time lapse.  There are several additional state-of-the-art features to this new system that have significantly improved the capabilities of the CTI.  The new system is equipped with software for:  1) fluorescence resonance energy transfer (FRET), a live-cell imaging technique used to determine the relative proximity of two molecules labeled with the appropriate FRET pair (YFP-CFP) via energy exchange; 2) fluorescence recovery after photobleaching (FRAP), a technique enabling dynamic studies of diffusional mobility and directed transport of free and coupled fluorophores in live cells; 3) fluorescence loss in photobleaching (FLIP), in which a single region of the cell is bleached continuously to assess the direction of intracellular trafficking by observation of a progressive loss of fluorescence moving from the bleached area in the direction of transport; and, 4) photoactivation of GFP or KAEDE.  It also has two metabolic modules, one for CO2 and another, newly introduced by Zeiss, for O2, plus a perfusion insert for live-cell imaging.  The microscope is mounted on a vibration-free isolation table.  Control and image acquisition and processing is through PC + LSM Software (v. 4.2).  A ReUse feature of the latter facilitates multi-user-friendliness by allowing the recall of all experimental parameters from stored images in each user’s archive, to instantaneously auto-set the whole system. The region of interest (ROI) capability, built into the software, allows ready focusing on a small region within the sample for precise pixel scanning and bleaching, essential for FRET, FRAP or FLIP measurements.  Multiple time-series capability allows for automated execution of complex time-series measurements, including motorized x/y stage for scanning multiple locations, with or without auto-focus, bleaching and triggering functions.  The whole system is protected by a GE 51002Y UPS power line conditioner/back-up system, to optimize performance and prevent loss of data during blackouts.

Nikon TE-300 Diaphot imaging system for fluorescence and DIC:  This system is designed for DIC imaging and fluorophore detection in fixed cells, as well as live cells in real time, and is additionally capable of ratiometric imaging.  The system consists of a Nikon Diaphot inverted microscope with high N/A, high magnification objectives, equipped for fluorescence and DIC, an X-Z stage controller, a heated stage, attached to a Sutter filter wheel containing multiple optical filters and an Orca digital camera.  Image acquisition and processing is through the MetaMorph workstation and software, attached to the microscope.

Stand-alone imaging workstation:  An additional workstation, supporting all imaging software of the systems above, is available in the Analytical, Metabolic, Instrumentation (AMI) Core, located in HMR-615, adjacent to the Cell and Tissue Imaging Core. It alleviates bottlenecks on the two microscopes by users with extensive post-acquisition image processing needs and thus frees the two microscopes above for their primary image-acquisition uses.  The workstation, based on a Dell Precision 670 system with a 30” screen, also facilitates on-line, local area network access to the users’ image and data files on the computers of the Zeiss LSM 510 and Nikon TE-300 Diaphot systems above. This facilitates remote access for processing and/or downloading of their image files, while the microscopes are occupied by others users.  The workstation supports offline versions of the software packages of the Zeiss LSM, MetaMorph, PCI C-Imaging, as well as other common, general-purpose imaging packages, such as Image J, Photoshop, etc. The users of the two microscopes are required and are responsible for downloading and backing up their files generated during microscopy sessions and removing them from hard drives of both systems’ within 2 weeks.

II. Instrumentation to be offered through facilitated/subsidized access to Center members (upon successful competitive renewal of RCLD grant):

GE DeltaVision OMX system:
http://cemma.usc.edu/instruments/

This system is located in the Center for Electron Microscopy and Microanalysis (CEMMA) at University Park Campus of USC, a 20 min shuttle ride for Health Sciences Campus users. The core is led by Mr. John Curulli.  The system includes an optical-sectioning epifluorescence microscope capable of operating in 3D-structured illumination microscopy or 3D-SIM, total internal reflection fluorescence (TIRF) microscopy or widefield-deconvolution mode.

Leica TCS SP5 Multiphoton system:

This system is housed in the Multi-Photon Microscopy Core at the USC Keck School of Medicine on the Health Sciences Campus, led by Dr. Janos Peti-Peterdi. The core is located in the Zilkha Neurogenetic Institute, ZNI332 and was established in 2008 as a result of a shared instrumentation grant (S10) from NIH.  The multi-photon Leica TCS SP5 fluorescence scanning confocal microscope is powered by a fully automated, broadly tunable (680-1080 nm) Ultra-II infrared pump laser (Coherent).

Perkin Elmer Ultraviewers 6 line Spinning Disk Laser Confocal Microscope:
http://uscnorriscancer.usc.edu/core/confocal/

This system is housed in the USC Norris Comprehensive Cancer Center and is part of the Cell and Tissue Imaging Core Facility maintained jointly by the Cancer Center and USC. The Core facility is led by Dr. David Hinton.  The spinning disk confocal microscope is an ultra-rapid confocal system for imaging live cells at high resolution, for long time-course experiments, and for multi-color fluorescent protein studies. It is capable of conducting high speed single or multiple probe time-lapse experiments.

Functions and Activities

The CTI Core provides the following functions and activities for Center members.  We will continue to allow limited access to non-members with NIH funding based on prior approval by RCLD’s Executive Committee to achieve Goals 1-3.  However, support for Goal 4 is to be a benefit solely for Center members and promising Junior Faculty.

Functions and Activities Supporting Goal 1:  To maintain the confocal and fluorescence microscopy facilities of RCLD.  The function of this Aim involves ensuring the CTI instruments are maintained in good working order under conditions ensuring the acquisition of reproducible results.  The activities include arrangements for regular service visits to ensure optimal alignment of optics and sample illumination.  As well, appropriate maintenance requires Ms. MacVeigh-Aloni, the Core’s research specialist, to be available, as she has routinely been, during assisted and independent imaging to ensure that the principles of appropriate usage are upheld and met. Maintenance requires coordination of instrument usage, an activity currently requiring manual or e-mail signup, but which will be accommodated by a new electronic signup system to be implemented in the new grant year, as well as distribution of information regarding changes in instrument availability, instrument repair, or updates in software to users, also coordinated through email and by postings on the Center’s website.  Training activities in Goals 2 and 3 overlap with this Aim by demonstrating safe and appropriate usage to enhance maximal accuracy, minimizing problems associated with instrument down time, and expanding the usage of core instrumentation by providing theoretical and practical workshops and hands-on training in more advanced capabilities such as fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET).

Functions and Activities Supporting Goal 2:  To provide assisted confocal and fluorescence microscopy and image processing to Center members.  The acquisition and analysis of imaging data from the Zeiss LSM 510 confocal and Nikon TE-300 Diaphot fluorescence microscopy-imaging systems with assistance from 50% effort Research Specialist, Ms. MacVeigh-Aloni, is the major activity supporting this Aim.  Ms. MacVeigh-Aloni operates the imaging equipment in collaboration with a member of the investigator’s laboratory, to aid in the identification of samples and images of interest.  However, experienced users who have been trained may also operate the imaging equipment relatively independently, with Ms. MacVeigh-Aloni available to address any technical issues that may arise.  Typically, about 50% of the RCLD CTI Core users utilize the instruments in an assisted imaging mode, while 50% use the instrumentation more independently. Imaging services routinely provided by Ms. MacVeigh-Aloni include standard confocal and fluorescence microscopy, real-time fluorescence and DIC imaging.

The usage of the more advanced capabilities of the Zeiss LSM 510 for FRET and FRAP have been less common by RCLD members.  In order to enhance such usage, Ms. MacVeigh-Aloni will work with Core Director, Dr. Hamm-Alvarez, to offer theoretical training regarding the potential applications for FRAP and FRET, and follow up by coordinating personal consultations, including advice on experimental design as well as assisted imaging in these techniques for interested RCLD members.

Ms. MacVeigh-Aloni is also responsible on a daily basis for monitoring and quality control of work, including trouble shooting for preparation of slides and image quality.  She is readily available to provide hands-on assistance and/or advice with sample preparation and microscope operation, in direct response to users’ concerns regarding quality control.

A major goal of the Core is education of its user base in all aspects of high-resolution imaging. One facet of this is the training by Ms. MacVeigh-Aloni for independent use of the Zeiss LSM 510 system. Some Core users, particularly graduate students and postdoctoral fellows, desire to master various imaging applications during the course of their research.  These users are trained by Ms. MacVeigh-Aloni on a one-on-one basis in their respective applications and, after vetting on the instrument, are allowed independent access.  Ms. MacVeigh-Aloni has attended a number of courses on computer graphics and advanced applications and is available to train as well as assist the users, as needed, in utilization of image analysis software tools to analyze features of their specimens.  She consistently assists RCLD Center members with preparation of publication quality graphical presentations of their images.  An additional stand-alone workstation, as mentioned above, is available with analysis software for both the confocal and fluorescence microscopes, plus other image processing software tools to Center members on a sign-up basis.

Functions and Activities Supporting Goal 3:  To provide education and training in diverse microscopy applications supported by the Core. In an effort to keep Center members regularly apprised of new imaging technologies applicable to their research projects, the Cell and Tissue Imaging Core will offer a regular series of imaging seminars by experts focused on the theory and practical applications of the imaging modalities available through the core:  laser scanning confocal fluorescence microscopy; FRAP, FRET, spinning disk confocal fluorescence microscopy; 3D-structured illumination microscopy (3D-SIM) and multiphoton fluorescence microscopy.  Dr. Hamm-Alvarez will work with Ms. MacVeigh-Aloni to enhance the theoretical and practical training regarding use of techniques such as FRAP and FRET for users, to be combined with practical and hands-on assisted imaging, using these techniques on the Zeiss LSM 510.  We anticipate that such training and practical assistance to increase the usage of this instrument as well as increase the sophistication of the usage applications.

For other new imaging modalities, which are to be introduced upon renewal of RCLD grant, appropriate faculty leadership from each core providing these new imaging services (Goal 4) will participate in seminars and directed workshops. As faculty directors, Drs. Janos Peti-Peterdi (Multiphoton Core) and David Hinton (Norris Cancer Center Cell and Tissue Imaging Core) will lead presentations on multiphoton fluorescence and spinning disk confocal fluorescence microscopy, respectively Scott Fraser, one of co-directors of the Center for Electron Microscopy and Microanalysis (CEMMA) Core, housing the 3D-SIM, will discuss the principles and applications of 3D-SIM to the Center membership These seminars will then be followed by group training sessions at the participating Core facilities by faculty and staff to enable experimental troubleshooting and planning for instrument use.  Examples of group training sessions that could be offered depending on interests include: 1) Use of the PerkinElmer spinning disk’s photokinesis unit for Fluorescence Recovery after Photobleaching (FRAP) and photo uncaging (Norris Cancer Center Cell and Tissue Imaging Core).  2) Environmental setup for live cell imaging, a live cell image acquisition demonstration, 3D reconstruction of the live cell image and the production of videos of live cells using spinning disk confocal fluorescence microscopy (Norris Cancer Center Cell and Tissue Imaging Core); and 3) Principles and operation of the OMX for 3D-SIM (CEMMA).  These workshops will provide special benefit to RCLD members because of the new agreements in place for member service as well as pilot funding support for RCLD member usage fees.

Additionally, as the University’s new Director of Science Initiatives and Director of the Translational Imaging Center, as well as co-faculty lead of the CEMMA Core, world-renowned microscopist Scott Fraser will also work closely with RCLD Center members to educate them regarding new pioneering technologies such as light sheet microscopy. New technologies will be made available to RCLD members as they are developed, and there is interest and need, similar to the current plan for pilot funding and access for RCLD members to University Park Campus’s 3D-SIM and Health Sciences Campus’s spinning disk confocal fluorescence and multiphoton fluorescence microscopes described below.  Guiding this development of the CTI Core, we will establish a CTI Internal Advisory Committee (CTI IAC), comprised of senior faculty imaging specialists (Fraser, Peti-Peterdi and Hinton) and liver researchers from the HSC, discussed more fully in Goal 4.

Functions and Activities supporting Goal 4:  To provide access and financial support for Center members to access USC Core imaging facilities enabling 3D-SIM, spinning disk confocal microscopy and multiphoton microscopy.  Three current core facilities exist at USC with the necessary instrumentation and expertise to facilitate Center members’ research interests in new modalities through access to 3D-SIM, multiphoton fluorescence microscopy and spinning disk confocal fluorescence microscopy.  Interest and need for these services among the membership was validated by a survey of all users of the Cell and Tissue Imaging Core, clearly indicating need for one or more advanced modalities across 70+% of our existing CTI RCLD member user base. A limiting factor in accessing state of the art imaging services is often a lack of financial resources enabling access to new technologies, which may or may not be yet grant-supported.  This situation is particularly acute for junior investigators, who represent a significant complement of our Core user base interested in more advanced imaging services.

Personnel of Cell and Tissue Imaging Core

Sarah F. Hamm-Alvarez, Ph.D., Director
Dr. Hamm-Alvarez (shalvar@pharmacy.usc.edu) is Gavin S. Herbert Professor of Pharmacology and Pharmaceutical Sciences (Pharmacy) and Professor of Physiology and Biophysics and Ophthalmology.  She also serves as Vice Dean for Research and Graduate Affairs and Executive Vice Dean at USC’s School of Pharmacy, and also as Director of Research Development for USC’s SC-CTSI.   She is a well-established cell biologist, working primarily on membrane trafficking processes facilitating regulated secretion of proteins through transcytotic and exocytotic mechanisms, as well as in endocytosis of a variety of macromolecules and ligands.  Her particular area of emphasis is the sorting, packaging and release of secretory proteins by acinar cells.  Although her major model system is the release of tear proteins from acinar cells of the lacrimal gland, she is considered an international expert in acinar cell biology and the biology/pathophysiology of exocrine glands which includes the exocrine pancreas and parotid gland, and she routinely collaborates and publishes work of impact to scientists working across the fields of exocrine gland biology. A significant recent emphasis of her work is eludication of the membrane trafficking mechanisms underlying and treatment of the autoimmune inflammation of exocrine glands associated with Sjögren’s syndrome.   A particular area of interest is the fidelity of sorting of lysosomal proteases such as cathepsin S from the trans-Golgi network to constitutive secretory vesicles versus a novel secretory lysosome population she has identified, and the dysregulation of this process in disease.  Her work, ranging from fundamental cell biology to animal to clinical studies, has led to the identification of putative tear biomarkers for identification of  Sjögren’s syndrome patients with early stage disease as well as the development of novel delivery strategies for targeting of immunomodulatory agents to lacrimal gland and ocular surface tissues and other sites of systemic inflammation. She also explores nanoparticle interactions with cells, specifically their mechanisms of internalization and intracellular sorting, in diverse cell types.  She has new collaborations in the area of Paneth cell development and function, based on her knowledge of mechanisms of exocytosis driven by diverse Rab proteins.  She has 25 years experience in various high-resolution light microscopy imaging applications and techniques, including confocal and multiphoton fluorescence microscopy and, more recently, 3D SIM.  She provides oversight for the Core including selection of seminar and workshop topics, coordination of pilot project selection and, of course, advice on experimental design and instrumentation usage as needed. Center members have been and will continue to meet with Dr. Hamm-Alvarez to discuss and explore the applications of imaging services to their specific needs.

Michelle MacVeigh-Aloni, B.S., M.S.  Research Specialist
Ms. MacVeigh-Aloni (macveigh@usc.edu) has been with RCLD since March 2000, initially as a full-time microscopy-imaging specialist, assisting our user base in all aspects of sample preparation and their confocal and fluorescence microscopy imaging needs. She comes with an extensive and excellent background, including over 30 years experience in electron and light microscopy, with 27 years of service at USC (since 1986).  Ms. MacVeigh-Aloni supervises the use of the Zeiss LSM 510 and Nikon imaging systems, providing assisted imaging and training, and will further work with the RCLD membership to provide new training in more advanced imaging capabilities including FRAP and FRET.

Management of the Cell and Tissue Imaging Core

Organization:

Formal announcements are the principal means by which the Core has and will periodically, as needed, communicate the availability of its on-going and newly added and/or upgraded resources and services. These are circulated widely via e-mail to all Center members and other investigators and will be posted on the Center’s website. Upon renewal of the Center grant in December 2015, notification of special imaging seminars and group training opportunities will also be distributed and posted as needed. The Core Director will work with the Executive Committee of RCLD to coordinate service offerings.  She will also work with the new CTI IAC, comprised of imaging experts and liver researchers, to evaluate the results of annual surveys regarding member service satisfaction and interests in new applications.  Finally, she will work with the CTI IAC to administer pilot funds for advanced services.

Prioritization of the use of the Subcore:

Priority for assisted and independent imaging with RCLD facilities is given to RCLD members, then non-members with NIH funding and finally other investigators. Potential candidates need to apply to the Center’s Executive Committee for approval of their membership under Organization). Day-to-day prioritization and potential conflicts of scheduling for access, if any, will be resolved by the Core Director in her periodic meeting with Ms. MacVeigh-Aloni.

Subsidized access through pilot funds to the additional facilities including 3D-SIM, spinning disk confocal fluorescence microscopy and multiphoton fluorescence microscopy will be for RCLD members.  As well, pilot funds will be awarded with a particular focus on assisting the many Junior Investigators with research goals in alignment with RCLD who are in need of access to these instruments, even if these investigators are not yet full members.  Needs of new and existing members will be evaluated by the Core Director, in concert with the Executive Committee to make sure that users are updated periodically, including investigators with need for advanced imaging capability.

Costs:

The Center grant covers salary + fringe of personnel. Thus, they do not contribute to cost bases for the Core users. These chargebacks are implemented on a two-tiered basis for the combined hourly usages of both systems, as follows:

Members: $25/hr with a total annual cap of $650
Non-members: $35/hr with a total annual cap of $1,300

In the new grant cycle, a new chargeback policy will be adopted.  We will work with the RCLD Executive Committee to evaluate the recovery at the end of the first year and, if necessary, will reduce or increase the charge-backs as required to generate the resources needed to continue to operate and support the Core’s instrument base, with a preferential support structure for the RCLD membership.

The pilot funds available for investigator access to the new imaging facilities described in Goal 4 will be provided by the School of Medicine with an annual contribution of $50,000 beginning in December 2015.

Benefits

The benefits of the Cell and Tissue Imaging Core are several-fold.  It provides essential services, in terms of state-of-the-art imaging, utilized by a large number of investigators, by offering access to shared resources and expert technical assistance at greatly reduced costs.  It also provides exceptional training opportunities for students and postdoctoral fellows in an area of research that is technologically innovative and cutting-edge.  Finally, due to the multi-user nature of the facility, it also provides an environment for collaboration and growth of investigators.  The Core Director, Dr. Hamm-Alvarez and its Research Specialist, Ms. MacVeigh-Aloni remain committed to providing state-of-the-art light microscopy imaging services and expertise to Center members through assisted imaging, as well as through periodic educational presentations, so that members may be kept abreast of new state-of-the-art technology and applications. As well, the Core Director is available to the members to consult on new imaging modalities and their potential applications to their research.

Recent Publications

The following are a few annotated examples of publications of Center members (highlighted in bold), which were especially enabled by the CTI Core:

  • Mo RZaro JOu J, Shen W.  Effects of Lipofectamine 2000/siRNA complexes on autophagy in hepatoma cells.  Mol Biotechnol.  51: 1-8, 2012.

    This important collaboration of two Center members used confocal microscopy to demonstrate that lipofectamine, a commonly used agent to deliver siRNA, increased autophagy in various hepatoma cell lines and everyone who uses such an approach needs to be aware of this.

  • Zhong S, Xu J, Li P, Tsukamoto H.  Caveosomal oxidative stress causes Src-p21ras activation and Lysine63 – TRAF6 protein polyubiquitination in iron-induced M1 hepatic macrophage activation.  J. Biol Chem. 287: 32078-32084, 2012.

    Confocal microscopy played a pivotal role in this collaborative study of Center members which demonstrates that endosomal Fe+2 – generated O2 activates hepatic M1 macrophages by inhibiting phosphotyrosine phosphatase (activating p21 and TAK1) and increasing Lys63 – polyUb of TRAF6 in caveosomes.

  • Chen W, Tseng C, Pfaffenbach K, Kanel G, Luo B, Stiles B, and Lee A.  Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis.  Hepatology 59:947-957, 2014.

    The collaboration of three Center members headed by Amy Lee was greatly facilitated by confocal microscopy of frozen liver tissue for co-localization of immunofluorescence as well as histology services including extensive analysis of routine H&E, TUNEL staining, Ki67 staining for proliferation, A6, α-smooth muscle actin, and others.

  • Chen WT, Tsukamoto H, Liu JC, Kashiwabara C, Feldman D, Sher L, Dooley S, Mishra L, Petrovic L, Hyeognam J and Machida K.  Reciprocal regulation by TLR4 oncogenic and TFF-β tumor suppressor pathways in liver tumor-initiating stemm-like cells.  J Clin Invest 123:2704-2717, 2013.

    These important collaborative findings suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Liver Histology (LH) Core

This Core was recently reorganized by splitting it from the former Cell and Tissue Imaging (CTI) Core, under which it was a subcore (the other being Microscopy subcore). Although the histology samples generated are often further evaluated in the adjacent Microscopy subcore (now named CTI Core), the chief Research Specialist of the Core, Ms. MacVeigh-Aloni, who manages and supports both Cores, assists in both services. The CTI and Liver Histology Cores provide distinct services and the Director of each has distinct expertise in liver histology and cell biology/imaging.

In 2014 Dr. Gary C. Kanel, a world-renowned expert with a 35-year career and background as a pathologist at the USC Liver Unit Laboratories, USC hospitals and LAC+USC Medical center, was appointed Director of the newly-organized Core.

See Prioritization of use of the Core section for the criteria and rules applying to the use of the Core. To access and use the Core contact Ms. MacVeigh-Aloni (macveigh@usc.edu), Research Specialist of the Core. Users are required to fill in and submit the Request for Histology Services form, along with delivering their tissue specimens to the Core at HMR-610A.

Goals

The long-term objectives of the Liver Histology Core are to:

  1. Perform routine histology, i.e. regular paraffin processing, sectioning and H&E, Sirius red and Oil red staining and provide assistance in interpretation and quantitation of findings
  2. Cut unstained slides, which the users can subsequently stain themselves
  3. Maintain a deparaffination and dehydration line of chemicals under a hood, accessible to users
  4. Provide frozen sectioning
  5. Add other services, based on the evolving needs of our user base (e.g. due to demand, over time the Core has added TUNEL, caspase 3, PCNA, BrdU, Grp78, Cox IV, F4/80, myeloperoxidase and Ki67 stainings
  6. Provide and train investigators in the use of a state-of-the-art laser micro-dissection system and customized slide preparations
  7. Provide a stand-alone workstation with microscope, image capture camera and processing software for independent and/or assisted use and analyses-interpretation of histology image
  8. Assist investigators in development of immunohistochemistry for specific needs
  9. Assist investigators in interpretation of histological findings and in the development of some quantitative and semi-quantitative systems for scoring findings (e.g. necrosis, inflammation, fibrosis, steatosis)

Facilities and Resources

Space:

Liver Histology Core is located on the 6th floor of the Hoffman Memorial Research (HMR) building in room HMR-610A, which houses its equipment base for sample preparation, processing, embedding, cutting and staining (see Equipment list below). Additionally, two of its equipment are installed in two adjacent labs, to accommodate better access for use. One is a cryostat (in HMR-612) and the other a laser microdissection system set up in the Analytical, Metabolic, Instrumentation (AMI) Core (HMR-615), where a stand-alone workstation is also set up for visualization, capture and processing of histology images.

Equipment:

The current equipment base consists of the following (with locations shown in parentheses):

  1. TBS ATP-120 Automatic Tissue Processor (HMR-610A)
  2. TBS Tissue Embedding Center (HMR-610A)
  3. Leica Autostainer XL (HMR-610A)
  4. Sakura Accu-cut SRM Microtome (HMR-610A)
  5. Leica Bond Max Immunostainer (HMR-610)
  6. Microm HM550 VP Cryostat (HMR-612)
  7. Leica LMD7000 laser micro-dissection system, complete with all modules, components and set of lenses (HMR-615). This state-of-the-art laser capture system is the most user-friendly among its peers, with an interface which allows users to begin utilizing the system after a 2-3 hour orientation and hands on training, with typically minimal technical assistance needed thereafter. The Core prepares all the frozen or other needed sections (5-200 micron thickness) on specialty slides for the users who subsequently perform their micro-dissections.
  8. Nikon 80i Eclipse microscope with Optronics MacroFire True Color 4MP (2048 x 2048P) digital CCD camera w/PictureFrame software (HMR-615). The system is interfaced with an image-processing Dell Precision 670 workstation with a 30-inch UltraSharp widescreen flat panel monitor. PictureFrame software facilitates the visualization, capture and subsequent processing of high-resolution digital images from histology slides. The workstation also supports various image processing-quantification software packages, including MetaMorph, Zeiss LSM 510, SimplePCI, plus general-purpose packages, such as Image J, Photoshop, etc. [Note: this system is shared with the Cell and Tissue Imaging (CTI) Core, sine it is configured for local area network access of image and data files on the computers of the Zeiss LSM 510 and the Nikon TE-300 Diaphot microscopy-imaging systems of the CTI Core, allowing users to download their image/data files remotely for their image processing-analysis needs while the microscopes are in use by others in their respective locations].

Functions and Activities

Services:

This Core was started as a subcore of the previous CTI Core in 2008 by offering regular paraffin processing, sectioning and H&E, Sirius and Oil Red staining, plus frozen sectioning. Over time, TUNEL staining was added, using the In Situ Cell Death Detection Kit, POD by Roche (Cat. No. 11 684 817 910), for applications requiring immunohistochemical detection and quantification of apoptosis through analysis by light microscopy. Thereafter, Caspase 3 detection was added and finally BrdU, PCNA, 3-Nitrotyrosine, Sirius Red, Oil Red O, etc. staining were added to the menu of the services offered (see request for services form). In addition, access to customized IHC stains developed for individual labs are available upon request: cytochrome c, cytochrome oxidase, RIPK1, Sab, myeloperoxidase, F4/80, P-JNK, nitrotyrosine, N-acetylated proteins, cyclin D, prohibitin.

Monitoring of Quality Control:

To provide quality assurance, adequate controls and normal tissue sections are used. In order to make sure that tissue sections are appropriate, the normal tissue sections (routinely stained with H&E) are used. Furthermore, for immunohistochemistry, several controls are applied, including negative controls (by omitting the primary antibody, for example). The other control is the positive control experimental samples, where positive result (staining) had been previously confirmed and the tested results can be compared with. When necessary, the tissue samples are stained in duplicates, in order to provide further stringent control and comparison. Dr. Kanel, Core Director, periodically reviews the stained slides to insure quality and interpretability. The Core retains unstained slides of liver tissue from various animal models that are used as positive controls for TUNEL, caspase 3, steatosis, fibrosis, BrdU and PCNA. These are stained at the same times as Center members’ experimental samples. In addition, Dr. Kanel can assist in interpretation of slides on an appointment basis (gkanel@dhs.lacounty.gov), which entails consultation with investigators and determination of the need to use grading scales or semi-quantitative image analysis, depending on the study.

Hands-on Use of Core Equipment:

The following items are offered for hands-on access/use:

  1. LMD7000 laser micro-dissection system. The latest (March 2012) and a most major addition to the Core during the current cycle was a state-of-the-art laser capture resource, i.e. Leica LMD7000 laser micro-dissection system. As stated, the distinction of this system is its utmost user-friendliness and ease of use, as compared to all counterparts, such that 2-3 hours of hands-on training and practice is sufficient to get the users going on their own. Nevertheless, Core’s Research Specialist Ms. MacVeigh-Aloni remains available and participates in many users’ sessions to teach and/or assist in their micro-dissection applications. The system is installed in the AMI Core (HMR-615), which also houses an Applied Biosystems/Life Technologies StepOnePlus real time PCR and LI-COR Odyssey 9120 Infrared Imaging systems. The former is essential for rapid and in tandem use with micro-dissections needing DNA/RNA analyses, which typically require hundreds of cells, while the latter is available for Western blot analyses, which typically require dissection of thousands of cells. The unique zonal architecture of the liver makes it an ideal organ/tissue for efficient and rapid micro-dissection of sufficient clusters and number of cells from its 3 zones for studies focusing on analysis of the distribution and presence-absence of the DNA/RNA and/or proteins of interest.
  2. Nikon 80i Eclipse microscope with Optronics MacroFire CCD camera and PictureFrame software interfaced with a Dell Precision 670 workstation supporting various image-processing software tools. An extensive base of investigators/labs use this system for observation, examination, processing and interpretation of their histology slides on their own, but frequently with assistance of the Core’s chief Research Specialist, Ms. MacVeigh-Aloni and, as needed, by Dr. Kanel, the Core Director.
  3. Among the rest of the Core’s equipment, the users are allowed hands-on access/use of only one item, i.e. the Leica Autostainer XL, to facilitate/expedite their work after hours or weekends, as needed.

Personnel of Liver Histology Core

Gary C. Kanel, M.D.  Director
Dr. Kanel (gkanel@dhs.lacounty.gov), Professor of Pathology, has been at Keck School of Medicine of USC since 1979. During 35 year career as a pathologist at the USC Liver Unit Laboratories, USC hospitals and LAC+USC Medical center, Dr. Kanel has written or co-authored 80 peer-review articles, almost all pertaining to liver diseases. Additionally he has written 4 books (first author in each) and is presently writing a 5th, all on the clinical, biologic behavior and pathology of a whole spectrum of liver diseases. He has also written 18 book chapters and 25 monographs and teaching materials. He has given over 100 lectures nationwide pertaining to liver pathology and has given over 25 sessions at the annual American Society of Clinical Pathologist meetings over the years. Dr. Kanel has extensive experience in the technical aspects of the preparation, staining and immunohistochemistry of liver. He will supervise the Core, assess the quality of the slide preparation, assist in troubleshooting and be available on a scheduled basis or by appointment to meet and review slides with Center members.

Michelle MacVeigh-Aloni, B.S., M.S.  Chief Research Specialist
Ms. MacVeigh-Aloni (macveigh@usc.edu) is an ASCP-certified Histotechnologist (1999) and Histology Technician (1992), who has had long-term and extensive experience and superb expertise in all aspects of histology and immunocytochemistry. The quality of her work is outstanding and highly valued and praised by investigators at USC, as well as many from other academic institutions engaged in collaborative work with our faculty investigators. Ms. MacVeigh-Aloni’s responsibilities include providing the full menu of the services listed on the Request for Histology Services form below, plus teaching-training and assistance in the use of the Core’s Leica LMD7000 laser micro-dissection system and the Nikon Eclipse 80i Microscope image capture and processing system for analyses and interpretation of histology slides. In addition, she is available to advise and assist investigators in the development of new IHC targets.

Heather Johnson, B.S.  Research Specialist
Ms. Johnson (heather.johnson@usc.edu) assists at 20% effort to provides support to the Core and Ms. MacVeigh-Aloni in all aspects of the Core’s services. She has been trained and teaches and assists investigators in the usage and applications of the core’s LMD7000 laser micro-dissection system, as well.

Management of Liver Histology Core

Organization:

Formal announcements are the principal means by which the Core has and will periodically, as needed, communicate the availability of its on-going and newly added and/or upgraded resources and services. These are circulated widely via e-mail to all Center members and other investigators and will be posted on the Center’s website. Customized special stains for individual Center members will get listed on our website to attract potential interest and collaboration of other Center members. Our users are encouraged and urged to preplan, project and communicate their needs to Ms. MacVeigh-Aloni to the extent possible to allow optimal allocation and utilization of the services and resources of the Core. Dr. Kanel will oversee the requests for services to insure that they are appropriate and consistent with the Center’s themes and overall aims.

Prioritization of use of the Core:

Priority for use of the Core is given to investigators involved in studying animal models of liver disease, followed by GI research. Center members receive first priority, with others accommodated based on availability of resources and technician time. Prospective users need to apply to the Executive Committee of RCLD for membership (see Membership and Qualification Criteria and Application for Center Membership under Organization), which in case of approval qualifies them as a priority user of the Core, or determines their non-member user status. Qualified users are investigators with peer-reviewed funding, as well as faculty with Center P/F Project or other start-up funds, or non-funded ones with approved protocols by the USC Animal Research Committee. Among them, priority is given to funded investigators and junior faculty. The Core services are geared to be essentially liver-specific, with few exceptions, which contributes to the very high efficiency of throughput and turn-around time, averting bottlenecks for Center members.Day-to-day prioritization is determined by Ms. MacVeigh-Aloni and, in case of development of significant bottlenecks, resolved by the Core Director. All users are required to show evidence of approval of protocols by Vertebrate Animal Committee. Routine human liver histology is not preformed at the Core. Specialized immunohistochemistry of human tissues can be performed in collaboration with Dr. Kanel in the hospital locations and under their billing arrangements, but is outside the scope of the RCLD Core.

Costs:

The Center grant covers 5% (salary+fringe) of the Director and 50% of the Chief Research Specialist. Thus, those items do not contribute to the cost bases and chargebacks for Core usage. At its inception in May 2007, the Core’s charges were set based on a survey of chargebacks of existing histology core facilities at USC. However, after the first year, they were adjusted downward to basically cover the costs of supplies and consumables. At the Core’s inception, all its equipment base was established through hand-me-down, used units, with no option to purchase service contracts for any. However, over time all units have been replaced through purchases of their advanced models, some brand new and others demo or refurbished sets, all with highly discounted extended multi-year warranty coverage, resulting in the Core’s charges remaining at highly competitive levels. Additionally, the continuous increase in the Core’s user base, including non-members, have resulted in the chargebacks generating sufficient funds, such that in the latest grant year it became possible to cover the cost of service contract of one major and critical item (i.e. Leica Bond Max Immunostainer). We will continue to review and may adjust our chargebacks upward, as needed, including higher rates for non-members, during the next cycle to generate sufficient funds to cover service contracts of the Core’s critical equipment, in case there may not be sufficient funds in the RCLD budget to cover the balance of the additional costs.

The fee schedule for our current menu of services appears on the Request for Histology Services form below. Our fee schedule remains highly competitive, as compared to other core services on campus, as well as outside services, since our Core staff salaries and equipment service contract and maintenance costs, with exception of one item (i.e. Leica Bond Max Immunostainer), are essentially all covered by the Center grant, resulting in significant savings to our investigators, especially junior faculty with start up and/or early grant funds. The fees have been established by monitoring and adjusting to the cost of supplies, the ongoing contract for the unit above (Leica Bond Max Immunostainer) and the share of personnel costs not covered by the Center grant. These services are aimed to provide cost savings to Center members, as well as to provide higher quality services than they could obtain elsewhere. As to the access/use of the LMD7000 laser capture system, it has been free of charge as of yet. However, we plan to have the regular users share 50% of the cost of its annual service contract.

Note: Users need to fill in and submit the Request for Histology Services form, along with delivering their tissue specimens to the Core at HMR-610A.

Analytical-Metabolic-Instrumentation (AMI) Core

Goals

The long-term objectives of the Liver Histology Core are to:

  1. Perform routine histology, i.e. regular paraffin processing, sectioning and H&E, Sirius red and Oil red staining and provide assistance in interpretation and quantitation of findings
  2. Cut unstained slides, which the users can subsequently stain themselves
  3. Maintain a deparaffination and dehydration line of chemicals under a hood, accessible to users
  4. Provide frozen sectioning
  5. Add other services, based on the evolving needs of our user base (e.g. due to demand, over time the Core has added TUNEL, caspase 3, PCNA, BrdU, Grp78, Cox IV, F4/80, myeloperoxidase and Ki67 stainings
  6. Provide and train investigators in the use of a state-of-the-art laser micro-dissection system and customized slide preparations
  7. Provide a stand-alone workstation with microscope, image capture camera and processing software for independent and/or assisted use and analyses-interpretation of histology image
  8. Assist investigators in development of immunohistochemistry for specific needs
  9. Assist investigators in interpretation of histological findings and in the development of some quantitative and semi-quantitative systems for scoring findings (e.g. necrosis, inflammation, fibrosis, steatosis)

Facilities and Resources

Space:

Liver Histology Core is located on the 6th floor of the Hoffman Memorial Research (HMR) building in room HMR-610A, which houses its equipment base for sample preparation, processing, embedding, cutting and staining (see Equipment list below). Additionally, two of its equipment are installed in two adjacent labs, to accommodate better access for use. One is a cryostat (in HMR-612) and the other a laser microdissection system set up in the Analytical, Metabolic, Instrumentation (AMI) Core (HMR-615), where a stand-alone workstation is also set up for visualization, capture and processing of histology images.

Equipment:

The current equipment base consists of the following (with locations shown in parentheses):

  1. TBS ATP-120 Automatic Tissue Processor (HMR-610A)
  2. TBS Tissue Embedding Center (HMR-610A)
  3. Leica Autostainer XL (HMR-610A)
  4. Sakura Accu-cut SRM Microtome (HMR-610A)
  5. Leica Bond Max Immunostainer (HMR-610)
  6. Microm HM550 VP Cryostat (HMR-612)
  7. Leica LMD7000 laser micro-dissection system, complete with all modules, components and set of lenses (HMR-615). This state-of-the-art laser capture system is the most user-friendly among its peers, with an interface which allows users to begin utilizing the system after a 2-3 hour orientation and hands on training, with typically minimal technical assistance needed thereafter. The Core prepares all the frozen or other needed sections (5-200 micron thickness) on specialty slides for the users who subsequently perform their micro-dissections.
  8. Nikon 80i Eclipse microscope with Optronics MacroFire True Color 4MP (2048 x 2048P) digital CCD camera w/PictureFrame software (HMR-615). The system is interfaced with an image-processing Dell Precision 670 workstation with a 30-inch UltraSharp widescreen flat panel monitor. PictureFrame software facilitates the visualization, capture and subsequent processing of high-resolution digital images from histology slides. The workstation also supports various image processing-quantification software packages, including MetaMorph, Zeiss LSM 510, SimplePCI, plus general-purpose packages, such as Image J, Photoshop, etc. [Note: this system is shared with the Cell and Tissue Imaging (CTI) Core, sine it is configured for local area network access of image and data files on the computers of the Zeiss LSM 510 and the Nikon TE-300 Diaphot microscopy-imaging systems of the CTI Core, allowing users to download their image/data files remotely for their image processing-analysis needs while the microscopes are in use by others in their respective locations].

Functions and Activities

Services:
This Core was started as a subcore of the previous CTI Core in 2008 by offering regular paraffin processing, sectioning and H&E, Sirius and Oil Red staining, plus frozen sectioning. Over time, TUNEL staining was added, using the In Situ Cell Death Detection Kit, POD by Roche (Cat. No. 11 684 817 910), for applications requiring immunohistochemical detection and quantification of apoptosis through analysis by light microscopy. Thereafter, Caspase 3 detection was added and finally BrdU, PCNA, 3-Nitrotyrosine, Sirius Red, Oil Red O, etc. staining were added to the menu of the services offered (see request for services form). In addition, access to customized IHC stains developed for individual labs are available upon request: cytochrome c, cytochrome oxidase, RIPK1, Sab, myeloperoxidase, F4/80, P-JNK, nitrotyrosine, N-acetylated proteins, cyclin D, prohibitin.

Monitoring of Quality Control:
To provide quality assurance, adequate controls and normal tissue sections are used. In order to make sure that tissue sections are appropriate, the normal tissue sections (routinely stained with H&E) are used. Furthermore, for immunohistochemistry, several controls are applied, including negative controls (by omitting the primary antibody, for example). The other control is the positive control experimental samples, where positive result (staining) had been previously confirmed and the tested results can be compared with. When necessary, the tissue samples are stained in duplicates, in order to provide further stringent control and comparison. Dr. Kanel, Core Director, periodically reviews the stained slides to insure quality and interpretability. The Core retains unstained slides of liver tissue from various animal models that are used as positive controls for TUNEL, caspase 3, steatosis, fibrosis, BrdU and PCNA. These are stained at the same times as Center members’ experimental samples. In addition, Dr. Kanel can assist in interpretation of slides on an appointment basis (gkanel@dhs.lacounty.gov), which entails consultation with investigators and determination of the need to use grading scales or semi-quantitative image analysis, depending on the study.

Hands-on Use of Core Equipment:

The following items are offered for hands-on access/use:

  1. LMD7000 laser micro-dissection system. The latest (March 2012) and a most major addition to the Core during the current cycle was a state-of-the-art laser capture resource, i.e. Leica LMD7000 laser micro-dissection system. As stated, the distinction of this system is its utmost user-friendliness and ease of use, as compared to all counterparts, such that 2-3 hours of hands-on training and practice is sufficient to get the users going on their own. Nevertheless, Core’s Research Specialist Ms. MacVeigh-Aloni remains available and participates in many users’ sessions to teach and/or assist in their micro-dissection applications. The system is installed in the AMI Core (HMR-615), which also houses an Applied Biosystems/Life Technologies StepOnePlus real time PCR and LI-COR Odyssey 9120 Infrared Imaging systems. The former is essential for rapid and in tandem use with micro-dissections needing DNA/RNA analyses, which typically require hundreds of cells, while the latter is available for Western blot analyses, which typically require dissection of thousands of cells. The unique zonal architecture of the liver makes it an ideal organ/tissue for efficient and rapid micro-dissection of sufficient clusters and number of cells from its 3 zones for studies focusing on analysis of the distribution and presence-absence of the DNA/RNA and/or proteins of interest.
  2. Nikon 80i Eclipse microscope with Optronics MacroFire CCD camera and PictureFrame software interfaced with a Dell Precision 670 workstation supporting various image-processing software tools. An extensive base of investigators/labs use this system for observation, examination, processing and interpretation of their histology slides on their own, but frequently with assistance of the Core’s chief Research Specialist, Ms. MacVeigh-Aloni and, as needed, by Dr. Kanel, the Core Director.
  3. Among the rest of the Core’s equipment, the users are allowed hands-on access/use of only one item, i.e. the Leica Autostainer XL, to facilitate/expedite their work after hours or weekends, as needed.

Personnel of Liver Histology Core

Gary C. Kanel, M.D.  Director
Dr. Kanel (gkanel@dhs.lacounty.gov), Professor of Pathology, has been at Keck School of Medicine of USC since 1979. During 35 year career as a pathologist at the USC Liver Unit Laboratories, USC hospitals and LAC+USC Medical center, Dr. Kanel has written or co-authored 80 peer-review articles, almost all pertaining to liver diseases. Additionally he has written 4 books (first author in each) and is presently writing a 5th, all on the clinical, biologic behavior and pathology of a whole spectrum of liver diseases. He has also written 18 book chapters and 25 monographs and teaching materials. He has given over 100 lectures nationwide pertaining to liver pathology and has given over 25 sessions at the annual American Society of Clinical Pathologist meetings over the years. Dr. Kanel has extensive experience in the technical aspects of the preparation, staining and immunohistochemistry of liver. He will supervise the Core, assess the quality of the slide preparation, assist in troubleshooting and be available on a scheduled basis or by appointment to meet and review slides with Center members.

Michelle MacVeigh-Aloni, B.S., M.S.  Chief Research Specialist
Ms. MacVeigh-Aloni (macveigh@usc.edu) is an ASCP-certified Histotechnologist (1999) and Histology Technician (1992), who has had long-term and extensive experience and superb expertise in all aspects of histology and immunocytochemistry. The quality of her work is outstanding and highly valued and praised by investigators at USC, as well as many from other academic institutions engaged in collaborative work with our faculty investigators. Ms. MacVeigh-Aloni’s responsibilities include providing the full menu of the services listed on the Request for Histology Services form below, plus teaching-training and assistance in the use of the Core’s Leica LMD7000 laser micro-dissection system and the Nikon Eclipse 80i Microscope image capture and processing system for analyses and interpretation of histology slides. In addition, she is available to advise and assist investigators in the development of new IHC targets.

Heather Johnson, B.S.  Research Specialist
Ms. Johnson (heather.johnson@usc.edu) assists at 20% effort to provides support to the Core and Ms. MacVeigh-Aloni in all aspects of the Core’s services. She has been trained and teaches and assists investigators in the usage and applications of the core’s LMD7000 laser micro-dissection system, as well.

Management of Liver Histology Core

Organization:

Formal announcements are the principal means by which the Core has and will periodically, as needed, communicate the availability of its on-going and newly added and/or upgraded resources and services. These are circulated widely via e-mail to all Center members and other investigators and will be posted on the Center’s website. Customized special stains for individual Center members will get listed on our website to attract potential interest and collaboration of other Center members. Our users are encouraged and urged to preplan, project and communicate their needs to Ms. MacVeigh-Aloni to the extent possible to allow optimal allocation and utilization of the services and resources of the Core. Dr. Kanel will oversee the requests for services to insure that they are appropriate and consistent with the Center’s themes and overall aims.

Prioritization of use of the Core:

Priority for use of the Core is given to investigators involved in studying animal models of liver disease, followed by GI research. Center members receive first priority, with others accommodated based on availability of resources and technician time. Prospective users need to apply to the Executive Committee of RCLD for membership (see Membership and Qualification Criteria and Application for Center Membership under Organization), which in case of approval qualifies them as a priority user of the Core, or determines their non-member user status. Qualified users are investigators with peer-reviewed funding, as well as faculty with Center P/F Project or other start-up funds, or non-funded ones with approved protocols by the USC Animal Research Committee. Among them, priority is given to funded investigators and junior faculty. The Core services are geared to be essentially liver-specific, with few exceptions, which contributes to the very high efficiency of throughput and turn-around time, averting bottlenecks for Center members.Day-to-day prioritization is determined by Ms. MacVeigh-Aloni and, in case of development of significant bottlenecks, resolved by the Core Director. All users are required to show evidence of approval of protocols by Vertebrate Animal Committee. Routine human liver histology is not preformed at the Core. Specialized immunohistochemistry of human tissues can be performed in collaboration with Dr. Kanel in the hospital locations and under their billing arrangements, but is outside the scope of the RCLD Core.

Costs:

The Center grant covers 5% (salary+fringe) of the Director and 50% of the Chief Research Specialist. Thus, those items do not contribute to the cost bases and chargebacks for Core usage. At its inception in May 2007, the Core’s charges were set based on a survey of chargebacks of existing histology core facilities at USC. However, after the first year, they were adjusted downward to basically cover the costs of supplies and consumables. At the Core’s inception, all its equipment base was established through hand-me-down, used units, with no option to purchase service contracts for any. However, over time all units have been replaced through purchases of their advanced models, some brand new and others demo or refurbished sets, all with highly discounted extended multi-year warranty coverage, resulting in the Core’s charges remaining at highly competitive levels. Additionally, the continuous increase in the Core’s user base, including non-members, have resulted in the chargebacks generating sufficient funds, such that in the latest grant year it became possible to cover the cost of service contract of one major and critical item (i.e. Leica Bond Max Immunostainer). We will continue to review and may adjust our chargebacks upward, as needed, including higher rates for non-members, during the next cycle to generate sufficient funds to cover service contracts of the Core’s critical equipment, in case there may not be sufficient funds in the RCLD budget to cover the balance of the additional costs.

The fee schedule for our current menu of services appears on the Request for Histology Services form below. Our fee schedule remains highly competitive, as compared to other core services on campus, as well as outside services, since our Core staff salaries and equipment service contract and maintenance costs, with exception of one item (i.e. Leica Bond Max Immunostainer), are essentially all covered by the Center grant, resulting in significant savings to our investigators, especially junior faculty with start up and/or early grant funds. The fees have been established by monitoring and adjusting to the cost of supplies, the ongoing contract for the unit above (Leica Bond Max Immunostainer) and the share of personnel costs not covered by the Center grant. These services are aimed to provide cost savings to Center members, as well as to provide higher quality services than they could obtain elsewhere. As to the access/use of the LMD7000 laser capture system, it has been free of charge as of yet. However, we plan to have the regular users share 50% of the cost of its annual service contract.

Note: Users need to fill in and submit the Request for Histology Services form, along with delivering their tissue specimens to the Core at HMR-610A.

Benefits

The Liver Histology Core provides both regular and specialized/customized services that have been in high demand by Center members. While some of the more routine services provided might be available through outside sources, they are much more costly, or not provided in a timely fashion to our user base.  All local and outside sources have much longer turnaround times, delaying the progress of the studies of our investigators. Having a well-organized, efficiently run Liver Histology Core in-house overcomes all of the above shortcomings-disadvantages, avoiding wasteful bottlenecks and set-backs. In addition, the Core remains highly flexible and responsive to the specific and continuously evolving needs of our investigators for customized applications. Furthermore, access of members to the expertise of Dr. Kanel, an established and superb liver pathologist, who assists in interpretation of histology results, is a major advantage not offered by other internal or external slide preparation services. As a sampling, the Core has assisted investigators in customized immunohistochemistry: P-JNK and Sab (Win), RIP 1 and 3 (Dara) cyclinD1 (Ji), prohibitin (Lu), PGAM5 (Kaplowitz), F4/80 and myeloperoxidase (Liu), cytochrome oxidase (Than), nitrotyrosine and N-acetylated proteins (Win/Than/Han/Kaplowitz). Also, many Center members use animal models of acute and chronic liver injury, fibrosis and liver cancer. Thus, having a core with special expertise in preparation of stains and interpretation of findings, as well as assistance in customized staining (e.g. IHC) is of great advantage to our community of liver investigators. Our membership will be made aware through our website of any specialized stains used by certain members so that they can communicate directly and develop new collaborations.

Recent Publications

The Liver Histology Core has been extensively utilized to support collaborative publications of Center members. An important feature is that the use often involved animal models of liver disease and the studies used nearly all of the Cores – e.g. cell culture of hepatocytes isolated from livers of disease models and controls by Cell Separation and Culture (CSC) Core, cells and liver sections being used for confocal microscopy and fluorescence imaging by Cell and Tissue Imaging (CTI) Core (e.g. apoptosis, necrosis, oxidative stress), histology and special stains for fat, apoptosis, proliferation, fibrosis by Liver Histology (LH) Core. In addition, secondary use of equipment and services of the Analytical, Metabolic, Instrumentation (AMI) Core frequently supported these studies, e.g. RT-PCR, ultracentrifugation for organelle fractionation, Odyssey imaging of Western blots, HPLC analyses, Seahorse oxygen consumption studies. Thus, the research of many Center members reflects integrated use of the four scientific Cores. A few representative examples of these are highlighted here (Center members in bold):

  • Chen W, Tseng C, Pfaffenbach K, Kanel G, Luo B, Stiles B, and Lee A.  Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis. Hepatology 2014 59: 947-57.

    The collaboration of three Center members headed by Amy Lee was greatly facilitated by histology services including extensive analysis of routine H&E, TUNEL staining, Ki67 staining for proliferation, A6, α-smooth muscle actin, and others.  Liver specific knockout of the ER chaperone, GRP94, led to hyperproliferation of periportal progenitor cells and deletion of both GRP94 and PTEN accelerated tumor formation preceded by disturbance of adhesion proteins and ERK activation (demonstrated on immunohistochemistry).

  • Ko K, Tomasi M, Iglesias-Ara A, French B, French S, Ramani K, Lozano R, Oh P, He L, Stiles B, Li T, Yang H, Martínez-Chantar M, Mato J, Lu S.

    Liver-Specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.  Hepatology 52: 2096-2108, 2010.  This collaborative group of Center members lead by Shelly Lu extensively utilized the histology core to study the consequences of liver specific knockout of prohibitin 1, which included apoptosis, proliferation, oxidative stress, fibrosis and malignant transformation.

  • Wang L, Wang X, Wang L, Chiu J, Van de Ven G, Gaarde W, and DeLeve L.  Hepatic vascular endothelial growth factor regulates recruitment of rat liver sinusoidal endothelial cell progenitor cells.  Gastroenterology 143: 1555-1563, 2013.

    This work extensively used the histology core services to demonstrate the key role of bone marrow derived liver sinusoidal endothelial progenitor cells in repopulating the liver sinusoidal cells following liver injury.

  • Win SThan TA, Han D, Petrovic LMKaplowitz N.  c-Jun N-terminal kinase (JNK)-dependent acute liver injury from acetaminophen or tumor necrosis factor (TNF) requires mitochondrial Sab protein expression in mice. J Biol Chem. 286: 35071-35078, 2011.

    This collaborative research of Center members was made possible by the use of the Liver Histology Core. H&E histology assessed liver injury and immunohistochemical determination of the zonal and subcellular distribution of protein nitrotyrosine formation and P-JNK was developed by the Core.  This work demonstrated that the interaction of P-JNK with mitochondrial Sab was required for necrosis due to acetaminophen and apoptosis due to TNF.

Collaborations

As seen above, a number of Center members collaborate with Dr. Kanel. This usually occurs when his participation is more extensive than his routine service provided to the Core. Also, as noted above, many of the publications, which used the Liver Histology Core (primary or secondary) were co-authored by multiple Center members. In addition, some of the special stains or immunohistochemistry developed for one Center member have lead to the utilization and participation of other members in collaborative research.

Furthermore, by listing on our website the specialized services developed in conjunction with individual investigators, we hope will encourage other Center members to develop collaborations with these investigators.

Central Core

The AMI Central Core is an essential and highly cost-effective resource for a large number of Center members and their labs, offering critically needed analytical services, provided through a versatile multi-detection HPLC system, plus an extensive base of common as well as advanced, state-of-the-art resources for shared access/use. The most critical and heavily used set of the Core’s resources is covered under service contracts, including preventive maintenance, which avert unexpected and wasteful downtimes. None of our Center members could independently equip their labs with the wide range and collection of resources the Central Core provides. The HPLC services, with continuously evolving-expanding menu of applications-compounds, is provided and performed hands-on by the Core’s Research Specialist, while the base of shared equipment is accessible on an on-demand hands-on basis to the users. A latest addition to the Core’s resources (see Equipment) has been a modeling-simulation workstation to support studies in drug induced liver injury (DILI) and other areas.

See Prioritization of use of the Core for the criteria and rules applying to the use of the Core. To access and use the Core, contact Dr. Ookhtens (murad.ookhtens@usc.edu), Core Director, or Ms. Heather Johnson (heather.johnson@usc.edu), Core’s Research Specialist.

Goals

The long-term objectives of the Central Core are to maintain and offer the following resources and services:

  1. An extensive base of critically needed common and advanced equipment for shared access-use
  2. A versatile menu of HPLC analyses and applications, using multiple detection methodologies
  3. A computer workstation for modeling-simulation and data analyses applications, currently offering DILIsym, MITOsym, SimPop and MATLAB software packages
  4. Assisting-advising investigators and their personnel in evaluation, selection and optimal utilization of the Cores base of resources and services
  5. Training-orientation of a constantly evolving base of users in hands-on operation of the Cores resources

Facilities and Resources – AMI Central Core

Space:

The Central Core occupies three laboratories, two on the 6th (400 and 120 sq. ft.) and one on the 8th (400 sq. ft.) floors of the Hoffman Memorial Research building (HMR), where a high concentration of Liver Center investigators are located.

Equipment:

The following is the base of equipment offered to RCLD members and other investigators on an on-demand and/or scheduled basis, with members receiving first priority. Location of each item on the 6th and 8th floors of the Hoffman Medical Research (HMR) building is shown in parentheses. All users and their lab personnel, including students, post-docs and fellows are trained by Dr. Ookhtens, Core Director, and Ms. Johnson, Core’s Research Specialist, and subsequently extended hands-on access to the equipment with the exception of the HPLC system. The latter is solely handled/operated by Ms. Johnson, with users procuring-providing their non-generic columns, mobile phases and samples in injection vials.

Note: The most critical and/or heavily used items (marked by * in the list below) are continuously covered under multi-year service contracts.

  1. * HPLC system: Shimadzu with dual LC-20AT pumps, SIL-20AC autosampler and CBM-20A controller, with following in-line detectors: SPD-M20A photodiode diode array (PDA); RF-10Axl fluorescence; ESA CoulArray 5600A electrochemical (ECD); IN/US b-RAM B radioactivity. The system is integrated with a HP Compaq dc5000 MT workstation, supporting control and data acquisition software, including Shimadzu LCsolutions, ESA CoulArray and Laura 4 packages. A 6-position FCV-20AH6 programmable column switcher facilitates ready switching between multiple applications (HMR-615).
  2. * Seahorse Bioscience Model XF24 Extracellular Flux Analyzer (HMR 612)
  3. * ABI/Life Technologies 7900HT Sequence Detection System for real time PCR + PC (HMR-615)
  4. * ABI/Life Technologies StepOne Plus 96-well RT PCR system w/PC control/data acq. (HMR-615)
  5. * ABI/Life Technologies Veriti Fast 96W Thermal Cycler w/PC control/data acquisition (HMR-615)
  6. * LI-COR Odyssey 9120 Infrared Imaging System w/PC control/data acquisition (HMR-615)
  7. * Thermo NanoDrop 8000 spectrophotometer w/PC control/data acquisition (HMR-615)
  8. * BMG FLUOstar Omega microplate reader w/PC control/data acquisition (HMR-615)
  9. Thermo Labsystems Luminoskan Ascent plate reader w/PC control/data acquisition (HMR-615)
  10. Shimadzu UV-2401PC spectrophotometer w/PC control/data acquisition + printer  (HMR-615)
  11. Beckman LS 6000TA scintillation spectrometer (HMR-610)
  12. Molecular Dynamics Storm 860 Phosphoimager w/PC & image analysis software (HMR-804)
  13. * Sorvall RC-6+ superspeed centrifuge with SS-34, GSA, SLA 1500 & FiberLite F13-14x50cy rotors (HMR-615)
  14. * Beckman L8-80M ultracentrifuge with 45Ti SW28 and SW41 rotors (HMR-610)
  15. Beckman Optima Max-E benchtop ultracentrifuge with TLA 100.3 rotor (HMR-610)
  16. Beckman Allegra 6R refrigerated benchtop centrifuge (HMR-610)
  17. Labnet HERMLE  Z400K refrigerated benchtop centrifuge (HMR-804)
  18. Fisher Scientific model 500 Sonic Dismembrator (HMR-804)
  19. COY Incubator w/CO2 & O2 control (614)
  20. COY Vacuum Airlock Anaerobic Chamber/Analyzer system (HMR-804)
  21. Vacuum Research Freeze Drier (804)
  22. Sanyo MDF-U71VC -86 degree freezer (HMR-805)
  23. VWR Symphony ULT -86 degree freezer (HMR-614)
  24. Kenmore -20 degree frost-free freezer (HMR-612)
  25. Millipore Milli-Q Advantage water purification system (HMR-614)
  26. Hoshizaki ice-maker (HMR-610)

Modeling-Simulation Workstation: A latest addition to the Core (2014) has been a powerful and versatile PC workstation to serve as a key resource to support modeling-simulation applications for current and future studies in drug induced liver injury (DILI) and other areas. The system currently supports DILIsym, MITOsym and SimPops, plus MATLAB software packages. MITOsym, developed very recently, is utilized as a powerful data analysis and modeling-simulation tool in tandem with studies using the Seahorse Extracellular Flux Analyzer.

Functions and Activities

The core offers an extensive base of equipment (see above) for shared access-use by Members, as well as other NIH-funded investigators, on an on-demand and/or sign-up bases, with Members receiving first priority. For HPLC services, users procure-provide all needed supplies-consumables, including non-generic columns, mobile phases and injection vials and submit their samples and mobile phases to the Core’s Research Specialist, who sets up, programs the runs and processes the chromatograms. This arrangement has served flawlessly over the years. Our versatile, multi-detector HPLC system was upgraded-expanded substantially at the end of previous cycle, by replacing many older modules by their up-to-date counterparts. The system is often utilized to set up, evaluate, cross-compare and modify-optimize an evolving-expanding menu of applications, listed in the table below.

Current menu of HPLC analysis offered based on the methods cited

HPLC_analysis.jpg

These methods have been used to analyze thiol-disulfides, GSH, GSSG, Cys (cysteine), Cys-Cys (cystine), Cys-SG (cysteine-glutathione mixed disulfides), cystathionine, homocysteine (Hcy), homocystine (Hcy-Hcy) and intermediates of trans-sulfuration pathway, SAMe (S-adenosyl methionine) and SAH (S-adenosyl homocysteine), as well as a variety of other compounds listed. The needs for HPLC applications and usage have come and gone in waves. In fact, during the period leading to our current resubmission, we have added and are evaluating-optimizing two new methods and for upcoming new projects to measure acetaminophen (APAP), its adducts and metabolites, and bilirubin.

Personnel of AMI Central Core

Murad Ookhtens, Ph.D.  Director
Dr. Ookhtens (murad.ookhtens@usc.edu) directs the core and manages/allocates optimal use and maintenance of its extensive shared instrumentation base, as well as its versatile, multi-detection HPLC system. He has over 40 years background as independent and collaborative investigator, with experience in myriad scientific applications, technologies and instrumentation. He has been involved with RCLD since its inception and has served as director and co-director of several of its evolving cores. He serves as the key person in acquisition of the extensive base of instrumentation for all RCLD cores, as well as GI/Liver Division’s research labs. Furthermore, as a bioengineer with extensive background, he is the key person to set up, expand and provide guidance and support for the Core’s current (DILIsym, MITOsym, SimPops and MATLAB) and future hardware-software tools for modeling-simulation applications. Dr. Ookhtens also oversees the Proteomics Subcore of the AMI Core and coordinates the tracking of its usage and chargeback processes.

Heather Johnson, B.S.  Research Specialist
Ms. Heather Johnson (heather.johnson@usc.edu) has received extensive training by Dr. Ookhtens and Mr. Kuhlenkamp, the Core’s previous Research Specialist, in the use and operation of the Core‘s HPLC system and the shared equipment base. She was recruited upon retirement of Mr. Kuhlenkamp in March 2012 and has been providing all HPLC services as well as assisting in the Core’s extensive instrumentation base’s day-to-day uses, operation, maintenance, repair, safety, plus all record-keeping and reporting of usages. A considerable amount of her effort is spent in orienting and instructing Center members and their laboratory staff in the operation and use of the shared equipment.

Management of AMI Central Core

Organization:

A description of the resources and services offered by the core is periodically updated to include-highlight any newly acquired resources and technologies and is distributed to Center members and posted on the Center’s website. To assist in optimal allocation and utilization of the resources and services of the Core, investigators are encouraged to pre-plan, project and communicate their needs on as long-term a basis as possible with the Core Director and Research Specialist.

Prioritization of use of the Core:

Qualified users are investigators with peer-reviewed funding, as well as faculty with Center P/F Project and junior faculty with start-up funds. Prospective users need to apply to the Executive Committee of RCLD for Membership (see Membership and Qualification Criteria and Application for Center Membership under Organization), which in case of approval qualifies them as priority user of the Core, while all others are accommodated based on availability of resources and technician time. For the latter, priority is given to funded investigators and junior faculty, with prioritization assigned by the Core Director. This applies to HPLC services, as well, which is restricted to Center members, since it involves allocation and commitment of the Core’s Research Specialist’s limited time. As to daily use of shared instruments, it is handled on a first-come first-served basis for items over which bottlenecks of access do not form. For the items that are in continuous high demand (e.g. RT-PCR systems) a sign-up for pre-scheduling is used.

Costs:

Based on the type and nature of resources and services provided, there has not been a need to implement any chargeback mechanism thus far. Center funds have been covering the costs of the most heavily used subset of equipment under annual service contracts, including preventive maintenance. Regarding HPLC services, as described under Functions and Activities above, the investigators procure and provide their non-generic specialty columns, plus all reagents/mobile phases and samples in injection vials to the Core Research Specialist, who sets up and programs the runs and processes-analyzes the chromatograms.

Benefits

The core maintains-offers an extensive and highly needed/used base of shared equipment, which lie outside affordability of acquisition and service-maintenance of most investigators’ budgets/labs, particularly junior faculty, who constitute a large base of the Core users. Thus, they benefit enormously from the ready access-use, as well as absence of time-consuming and costly downtimes and maintenance costs. Also, they benefit from training in the use of all equipment offered by the Core. The Core develops and assists in development of new, efficient and cost-effective methodologies and assays, replacing existing ones, which reduce the investigators’ cost bases (e.g. adaptation of an assay to measure ALT, frequently needed by Center member investigators, to a micro-plate reader, reducing the cost per sample from $7.50, charged by clinical labs, to a few cents). These assays are taught to the members and their laboratory staff, who then conduct them on their own, using the Core’s equipment. In addition, the Core offers-maintains certain state-of-the art, advanced and/or specialty resources, essential to the work of some investigators and projects underway. An example is the Seahorse XF24 Extracellular Flux Analyzer, acquired to support ongoing major and critical studies focused on hepatic drug and fatty acid toxicity and the critical role of mitochondrial respiration. Finally, the core provides HPLC services, conducted hands-on by its Research Specialist, who keeps up-to-date with all aspects of past and new applications, plus troubleshooting-maintenance skills (through vendor workshops). Thus, the resources and expertise offered by the core facilitates the work of a large base of our investigators, contributing to highly efficient and cost-effective progress of their work. Finally, the core serves as a critical resource to test and develop new analytical approaches and methods.

Recent Publications(members in bold)

The following collaborative publications used HPLC analyses provided by the Core:

  1. Garcia J, Han D, Sancheti H, Yap LP, Kaplowitz N and E Cadenas. Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates. J Biol Chem. 2010 Dec 17; 285(51): 39646-54. PMID: 20937819. PMCID: PMC3000945.
  2. Li J, Ramani K, Sun Z, Zee C, Grant EG, Yang HP, Xia M, Oh P, Ko K, Mato JM and SC Lu. Forced Expression of Methionine Adenosyltransferase 1A in Human Hepatoma Cells Suppresses in vivo Tumorigenicity in Mice. Am J Pathol. 2010 May; 176(5): 2456-2466. Li J and Ramani K share equal authorship. PMID: 20363925. PMCID: PMC2861110.
  3. Ramani K, Yang HP, Kuhlenkamp J, Tomasi L, Tsukamoto H, Mato JM and SC Lu. Changes in the Expression of Methionine Adenosyltransferase Genes and S-adenosylmethionine Homeostasis during Hepatic Stellate Cell Activation. Hepatology. 2010 Mar; 51(3): 986-995. PMID: 20043323. PMCID: PMC2905860.
  4. Shinohara M, Ji C and N Kaplowitz. Differences in betaine-homocysteine methyltransferase expression, endoplasmic reticulum stress response, and liver injury between alcohol-fed mice and rats. Hepatology. 2010 Mar; 51(3):796-805. PMID: 20069651. PMCID: PMC2840074.
  5. Shinohara M, Ybanez MD, Win S, Than TA, Jain S, Gaarde WA, Han D and N Kaplowitz. Silencing glycogen synthase kinase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1. J Biol Chem. 2010 Mar 12; 285(11): 8244-55. PMID: 20061376. PMCID: PMC2832976
  6. Tomasi ML, Ramani K, Li T, Ko K, Yang HP, Bardag-Gorce F, Ara A, Feo F, Pascale M, Mato JM and SC Lu. S-Adenosylmethionine Regulates Dual-Specificity MAPK Phosphatase Expression in Mouse and Human Hepatocytes. Hepatology. 2010 Jun; 51(6): 2152-61. PMID: 20196119. PMCID: PMC2905543.
  7. Han D, Ybanez MD, Johnson HS, McDonald JN, Mesropyan L, Sancheti H, Martin G, Martin A, Lim AM, Dara L, Cadenas E, Tsukamoto H and N Kaplowitz. Dynamic adaptation of liver mitochondria to chronic alcohol feeding in mice: biogenesis, remodeling, and functional alterations. J Biol Chem. 2012 Dec 7; 287(50): 42165-79. PMID: 23086958. PMCID: PMC3516762.
  8. Kao E, Shinohara M, Feng M, Lau MY and C Ji. Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes. Hepatology. 2012 Aug; 56(2):594-604. PMID: 22407670. PMCID: PMC3406240.
  9. Li TWH, Yang HP, Peng H, Xia M, Mato JM, and SC Lu. Effects of S-adenosylmethionine and methylthioadenosine on inflammation-induced colon cancer in mice. Carcinogenesis. 2012 Feb; 33(2): 427-435. PMID: 22159228. PMCID: PMC3279046.
  10. Tomasi ML, Ryoo M, Skay A, Tomasi I, Giordano P, Mato JM and SC Lu. Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer. Exp Cell Res. 2013 Jul 15; 319(12):1902-11. PMID: 23588207. PMCID: PMC3700574.
  11. Yang HP, Cho ME, Li TWH, Peng H, Ko KS, Mato JM and SC Lu.  MiRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma. J Clin Invest. 2013 Jan 2; 123(1):285-298. PMID: 23241961. PMCID: PMC3533284
  12. Chang AH, Sancheti H, Garcia J, Kaplowitz N, Cadenas E and D Han.  Respiratory substrates regulate S-nitrosylation of mitochondrial proteins through a thiol-dependent pathway. Chem Res Toxicol. 2014 May 19; 27(5): 794-804. doi: 10.1021/tx400462r. Epub 2014 Apr 23. PMID: 24716714. PMCID: PMC4033640.
  13. Saberi B, Ybanez MD, Johnson HS, Gaarde WA, Han D and N Kaplowitz. Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways. Hepatology. 2014 Apr; 59(4): 1543-54. PMID: 23873604. PMCID: PMC3997165 [Available on 2015/4/1].

Proteomics Subcore

This Subcore was established under the newly structured Analytical, Metabolic, Instrumentation Core of the Research Center for Liver Diseases (RCLD) and announced in November 2007 to the RCLD members, which was the exclusive user base it was called to serve. Initially, the Subcore was in Dr. Zandi’s laboratory and was equipped with only one LTQ mass spectrometer.  As mass spectrometry and other technologies for proteomics kept advancing rapidly, and the cost of new instruments were quite high and not within the means of any single laboratory, Dr. Zandi proposed the establishment of a university-wide Proteomics Core at USC. The office of the Provost provided the capital funding for new instrument purchases and space renovation for a ground-up proteomics core and Keck School of Medicine of USC and RCLD provided support for operating costs. The office of the Provost also provided for instrument upgrades. This upgraded an LTQ XL instrument to a new Orbitrap. The Proteomics Core has recently installed a new Q Exactive MS with funds provided by USC’s Office of Research, and the Dean’s Offices of Keck Schools of Medicine and Pharmacy (see Proteomics Core of USC) The Proteomics Subore has developed specific protein isolation and quantification methods for RCLD members. Drs. Zandi, Subcore Director, and Zhou, Subcore Manager, have been providing advice to many RCLD members for their proteomics projects on an ongoing basis.

See Prioritization of use of the Subore for the criteria and rules applying to its use. To use the services of the Subcore, contact Dr. Zandi (zandi@usc.edu), Director, or Dr. Zhou (zhou287@usc.edu), Manager.

Goals

The objectives of the Proteomics Subcore are to provide state-of-the-art mass spectrometry-based proteomics services including:

  1. Protein sequencing and identification from gel bands and complex mixture of proteins in solutions
  2. Analysis and determination of post-translational modification in proteins
  3. Quantitative and qualitative proteomic profiling
  4. Advice/assist with experimental strategies-designs related to protein purification and proteomics projects.

Facilities and Resources

The Proteomics Subcore has been providing services to RCLD members for mass spectrometry-based single and complex mixture protein sequencing, identification, quantification and PTM analysis. It has also provided consultations and guidance, and developed methodologies for sample preparations, immuno-precipitation of protein complexes, and direct identification of immune-complexes. The Subcore is a university wide core, partially supported by the RCLD.

Space:

The Proteomics Core of USC is located on the 5th floor in the Hoffman Medical Research Building (HMR) rooms 511 and 513 in a 1000 sq ft of newly renovated laboratory space designed for proteomics.  Thus, it is one floor below, directly under the Central Core. A large number of RCLD members and its administrative office are located on various floors in HMR and adjacent buildings.

Equipment:

The following equipment are available for protein identification in the Proteomics Core. They are operated by Dr. Zhou, Core Manager, and Dr. Zandi, Core Director, and are made available to RCLD members on a priority basis under a Subcore structure.

  1. Q Exactive Hybrid Mass Spectrometer
    The Q Exactive is a high performance benchtop quadrupole Orbitrap system based on the combination of the most advanced quadrupole technology combined with Orbitrap analyzer technique. Implementation of a quadrupole mass filter in front of the curved ion trap (C-Trap) allows for precursor ion selection and hence for MS/MS and SIM scan modes besides the ability of full MS mode. Together with the new and unique scan type of spectra multiplexing, where multiple preselected precursors are collected in the C-Trap for simultaneous high resolution detection in the Orbitrap further reduction of cycle time can be accomplished. Q Exactive is the most sensitive bench-top quadrupole high resolution mass spectrometer perfectly combining precise quantification and high performance qualitative work in one instrument.
  2. EASY-nLCTM 1000 Integrated Ultra High Pressure Nano-HPLC System.
    The EASY-nLC 1000 is a split-free, nano-flow liquid chromatograph optimized for separating biomolecules such as proteins and peptides at ultra high pressures up to 1000 bar or 15,000 psi. EASY-nLC 1000 is a fully integrated LC-system with a binary nano-flow gradient pump, a cooled autosampler, switching valves and high precision flow sensors for accurate solvent control before high-pressure mixing.
  3. LTQ Orbitrap ETD Hybrid Mass Spectrometer
    The Thermo Electron Finnigan LTQ Orbitrap hybrid mass spectrometer is a linear ion trap mass spectrometer equipped with an electrospray ionization source. The LTQ Orbitrap is a state-of-the-art mass spectrometer with improved capacity, trapping efficiency, scan speed, and high mass accuracy and resolution. Electron Transfer Dissociation (ETD) enables peptide dissociation by transferring electrons to positively charged peptides, leading to a rich ladder of sequence ions derived from cleavage at the amide groups along the peptide backbone. Amino acid side chains and important modifications such as phosphorylation are preserved intact and can be readily assigned as a result of analysis. Since large peptides can be successfully fragmented by ETD, this enables top-down and middle-down sequencing of small proteins/large peptides and supports bottom-up proteomics using enzymes that make larger peptide fragments.
  4. Eksigent NanoLC-2D
    The Eksigent NanoLC-2D is a fully automated 2-dimensional chromatography system ideal for whole proteome profiling, low abundance protein identification, and post translational modification. Built with a 10-port column switching valve, 2 binary gradient pumps and auto-sampler, the NanoLC-2D system does not only provide high-throughput proteomics, it is designed for flexibility.
  5. Autosampler
  6. Computers: Four computers with Proteome Discoverer 1.3 and 1.4 softwares for instrument operation and data analysis.
  7. Pressure Injection Platform: The high-pressure injection “bomb” is used for sample loading and micro-column preparations.
  8. Micropipette Laser Puller Model P-2000 (Sutter Instrument Co.): The laser puller is used to prepare spray tips for the micro-columns (10 cm x 75 micron ID) for direct spraying of the column.
  9. 2D Gel apparatus
  10. In addition to the large equipment listed above, the core laboratory is equipped with all necessary small equipment such as table top centrifuges, Speed vac, heat blocks, deep freezer, etc. for sample preparation, handling, and storage.
  11. Furthermore, the core fabricates and packs chromatography micro-columns for nano-HPLC separations of peptides for in-line 1D and 2D LC/MS/MS analysis. These include the spray tips fabricated from fused silica and packed with different chromatography resins. Dr. Zandi has developed column systems for two-dimensional chromatography (Wang et al, 2008). These result in significant savings, providing the highest quality and sensitivity protein analytic services at very low costs and reduced charge structure.

Functions and Activities

Services:

The Proteomics Subcore is a fee for services core, and the following services in this renewal proposal are currently provided and will continue to be provided.

a) Consultation with Dr. Zandi (free of charge for RCLD members). Initially, Dr. Zandi and RCLD members discuss the objectives of their research studies and how techniques offered by the Proteomics Subcore could assist the Center members’ research program.  Dr. Zandi’s combined expertise in biochemistry, cell signaling, molecular biology, and proteomics puts him in a unique position to better understand the biological projects of RCLD members and offer advice as to how proteomics technologies can be used for each project.

b) List of specific services offered by Proteomics Subcore.

i) improved protein identification for qualitative and quantitative shotgun proteomics analysis.
To overcome the most common bottleneck of obtaining deep proteome coverage from complex mixtures, we have developed two strategies in the Proteomics Subcore (Figure 1).

Figure 1. Layout of Experimental Design combining HILIC/SCX pre-fractionation with ELC.

First, since ample proteins are available in most shotgun proteomics, we have used two complementary fractionations in the first dimension separation of peptides, Strong Cation Exchange (SCX) and Hydrophilic interaction (HILC). Second, we have scripted and used an Exclusion List Convertor (ELC) method (Zhou and Zandi, manuscript submitted) to increase the number of peptides identified per sample. Using these strategies we have increased the number of proteins identified in pediatric fecal bacterial samples from patients with Ulcerative Colitis by about 10-fold (Figure 2).

Figure 2. Using ELC increases identified bacterial proteins by 10-folds. The strategy outlined in Figure 1 was used to compare ELC to standard DDA (data dependent acquisition) mass spectrometry methods.

The experimental layout shown in Figure 1 can be adapted for in vitro labeling of peptides, for example using TMT tags for multiplex analysis of protein mixtures as shown in Figure 3.

Figure 3. An example of a layout of experimental design for a quantitative TMT tag labeling for three protein mixtures.

ii) Mass Spectrometry- based analysis of immune-complexes on the beads.
One of the most utilized methods to identify interacting proteins and protein complexes is the immuno-precipitation (IP) by protein specific antibodies followed by MS-based protein identification from SDS-PAGE gel bands, or eluted proteins. The latter cases are both inefficient, and many low abundant interacting proteins are not identified in SDS- PAGE (due to lack of sensitivity of gel staining methods), or lost during elution of immune complexes. To overcome these shortcomings, the core developed a method to directly proteolyse proteins off the beads after IP. Figure 4, (Left side) shows an outline of the experimental strategy combined with TMT mass tag labeling and profiling of two complex mixtures, after immune-precipitation using control and specific antibodies. This method was used to compare protein complexes of Sab protein from the liver mitochondrial proteome from mice untreated or treated with APAP (Figure 4, right), and is currently being used by RCLD members.

Figure 4. Left: Comparative protein profiling of two complex protein mixtures using immunoprecipitation in combination with TMT quantitative proteins. Right: Venn diagram shows endogenous Sab interacting proteins, using strategy described on the left. Con, control IgG; Sab; Sab antibody; NT, untreated. Sab was Immuno-precipitated from mitochondrial protein extracts livers of untreated or acetaminophen treated mice.

iii) Sequencing and identification of proteins from gel bands and mixture of proteins in solution by mass spectrometry. The Subcore provides protein sequencing and identification from 1-D and 2-D gel bands.  For sequencing and identification of proteins in complex mixtures (shotgun approach), the Subcore has developed novel strategies to increase the number and the coverage of identified proteins. This approach can be used for label-free, as well as quantitative proteomics, where peptides are labeled with a variety of stable isotopes (in vivo), or TMT or iTRAQ (in vitro).

iv) Post-Translational Modification (PTM) enrichment and determination. Identification of specific PTM sites, such as phosphorylation, ubiquitination, glycosylation, and methylation, occurring in proteins are best achieved after peptides containing such PTMs are enriched. The Subcore will use established methods and PTM-specific enrichment commercial kits (for example, Pierce Phosphoprotein, Glycoprotein, and Ubiquitin Enrichment kits) to facilitate identification and determination of PTMs for RCLD members.

v) Quantitative Protein Profiling using in vitro labeling with TMT Tags. Quantitative measurement of changes in protein expression and post-translational modifications are critical in understanding the mechanisms of biochemical processes underlying healthy and disease conditions. Tandem Mass Tag Technology is a method that enables concurrent identification and quantification of proteins in different samples by tandem mass spectrometry. In this method the amino termini of lysine residues of peptides are modified with small isobaric molecules. These molecules have identical chemical structures, but contain different amounts of heavy 13C isotopes. Thus, they have different molecular weights, which are detected and quantified by mass spectrometer. This method is advantageous when in vivo isotopic labeling is not feasible. With this method eight different samples can be labeled and analyzed simultaneously for quantitative comparison. Reagents for Tandem Mass Tags are available through Thermo Scientific. Proteins can also be labeled in vivo using amino acids containing stable isotopes (13C or 15N).  In vivo isotope labeling and protein preparation procedures will be carried out by investigators with consultation with Drs. Zandi and Zhou.

vi) Intact protein analysis. The Orbitrap mass spectrometers are capable of intact protein analysis. The Subcore will provide this service to RCLD members. Users will purify the target protein, and the Subcore will analyze the intact protein mass.

c) Informatics support. The Subcore provides the necessary informatics support, such as data base searches, peptide matching and identification, PTM site identification and quantification, and comparative qualitative and quantitative data analysis for all the mass spectrometry-based services listed above.

d) Development of additional services
The field of proteomics is expanding rapidly. The Subcore will have educational seminars to inform RCLD investigators of the developments of new proteomics approaches. Based on the need and interest of the investigators, the Subcore will incorporate new services.

Monitoring of Quality Control:

Two levels of quality control for MS-based protein identification are applied in the Subcore. First, instruments are under service contracts and their functionality is tested during maintenance visits as needed, and by core staff by using calibration masses as recommended by the vendor on a weekly basis. Second, the nano liquid chromatography (LC) of peptides in combination with MS functions is tested by using a standard digested BSA protein before and after each LC/MS run. Routinely, we achieve 80% protein coverage from one pico mol of digested BSA. This indicates proper functioning of all instruments, the steps of sample handling, as well as data base search and peptides matching.

Personnel of Proteomics Subcore

Ebrahim Zandi , Ph.D.  Subcore Director
Dr. Zandi (zandi@usc.edu) is founder of the Proteomics Core at USC and has served as its Director since its inception. Initially he established a biological mass spectrometry-based proteomic program in his group in 2005. On the basis of the expertise of his group, he then initiated the establishment of the university-wide Proteomics Core facility at USC. Dr. Zandi is a biochemist and molecular biologist with expertise in mechanisms of signal transduction of pro-inflammatory pathways, such as TNF and IL-1 signaling. He has been a primary faculty member in the department of Molecular Microbiology and Immunology at the Keck School of Medicine since 1999.

Yu Zhou, Ph.D.   Subcore Manager
Dr. Zhou (zhou287@usc.edu) received her Ph.D. in Analytical Chemistry from Peking University. She was a group leader and Senior Research Scientist in R&D in Changsha Cigarette Factory in China, where she used various mass spectrometry and analytical methods. In 2008 she joined the National Institute on Aging at NIH as a postdoctoral fellow. At NIH she developed protein purification methods for mass spectrometry and proteomics, and analyzed post-translational modifications (PTMs). Dr. Zhou is highly skilled in proteomics technologies and has published 26 papers in the field.

Management of Proteomics Subcore

Organization:

Dr. Zandi serves as the Subcore director and supervises Dr. Zhou, the Subcore manager. Dr. Zandi oversees all the operations of the core. Dr. Zhou is in charge of sample preparation, instrument operation, and data analysis. RCLD provides administrative support by placing orders for supplies and other items needed for the Subcore, and maintains the charge back system for the RCLD members.

Prioritization of use of the Subcore:

The Proteomics Core is a university-wide core. However, since RCLD directly contributes to the operating budget of the core, full time RCLD members have priority to use the services, and receive a 10% discount. Drs. Zandi and Zhou provide consultations for proteomics projects and study design for all RCLD members free of charge. Prospective users need to apply to the Executive Committee of RCLD for Membership (see Membership and Qualification Criteria and Application for Center Membership under Organization). Prioritization of services for RCLD members is determined by reviewing the number of requests and allowing only one project per investigator to encourage use of this service by many Center members. In cases in which multiple requests are received, the Executive Committee needs to review the request and prioritize the services based on research program of Center members and relevance to the Center’s themes.

Costs:

Cost for services provided by the Proteomic Subcore are based on the use of disposable items, prorated expenses of operating the MS/MS and providing partial support for service contracts on these highly sophisticated equipment items. In addition to the necessary MS equipment, the Subcore fabricates and packs chromatography micro-columns for nano-HPLC separations of peptides for in-line 1D and 2D LC/MS/MS analysis. These include the spray tips fabricated from fused silica and packed with different chromatography resins. Services and cost comparisons (Members vs. non-Members) are listed below. The fees cover in part the cost of sample preparation supplies, such as HPLC-grade solvents, reagents, tubes, proteolytic enzymes, micro-column chromatography fabrication material, instrument maintenance, and annual service contracts.

All prices include: A Quality Control run (BSA standard 100 fmol), Proteolytic Digestion and desalting, LC-MS/MS analysis, Database search, informatics and bioinformatics analysis as needed.

  1. Protein Identification: a protein or proteins from a gel band (coomassie or silver stained), or in solution.
    Unit Price: RCLD Members $180, KSOM $200, USC (not KSOM) $300, Outside of USC $400.
  2. Post-Translational Modification (phosphorylation, ubiquitination, acetylation, methylation, etc.) discovery without PTM enrichment.
  3. Post Translational Modification Discovery with PTM enrichment.
    Unit Price: RCLD Members $900, KSOM $1000, USC (not KSOM) $1500, Outside of USC $2000.
  4. Quantitative proteomics using mass tags such as TMT, iTRAQ, SILAC, or label-free.
    Unit Price: RCLD Members $2700, KSOM $3000, USC (not KSOM) $4500, Outside of USC $6000
  5. Custom and non-standard proteomics analyses. Examples of services that have been provided in the past funding period: Protein-protein interaction, Protein-drug interaction, and Targeted Proteomics.

Unit Price: The cost of each service is determined based on the type of the material required and effort level needed for the experiments.

In addition, for each of the services outlined above, the cost will also be reviewed on an individual basis, if unique reagents are used, or there is a need for additional runs to generate peptide sequences to adequately identify proteins contained within the mixture. Costs of studies for quantitative proteomics will be individually assessed depending on the complexity of protein mixtures, and number of times the sample needs to be processed by the LCMS/MS instrument in order to sequence sufficient regions of the protein to adequately identify it. Dr. Zandi will oversee the evaluation of the MS/MS data, and determine if samples need to be analyzed on the MS/MS instrument to provide additional peptide sequence to adequately identify the protein.

Benefits

Proteomics technologies require expensive equipment and a high degree of expertise and experience in at least three different fields including mass spectrometry, peptide and protein separation technologies, and bioinformatics. Obviously it is not possible for each laboratory to acquire such expensive instrumentation and expertise. Therefore, having the Proteomics Subcore in house is of great benefit to all investigators. An additional benefit of this Subcore is the dual expertise of Subcore Director Dr. Zandi. He is a biochemist and molecular biologist, who has also become an expert in proteomics. This is of great benefit to RCLD members, as Dr. Zandi’s knowledge and understanding of biological studies puts him in a unique position to provide the best advice for the use of proteomics technology.

Recent Publications
(members in bold)

The following publications were made possible by use of the Proteomics Subcore services, which included mass spectrometry-based protein identification,  phosphorylation site identification, and drug protein interaction site mapping.

  1. Xia M, Chen Y, Wang LC, Zandi E, Yang HP, Bemanian S, Martínez-Chantar ML, Mato JM, Lu SC. Novel function and intracellular localization of methionine adenosyltransferase 2beta splicing variants. J Biol Chem.  2010, 285:20015-20021. PMCID: 20421296.

    Human methionine adenosyltransferase 2beta (MAT2beta) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The Proteomics Subcore provided advice for protein pull down strategies for V1 and V2 and sequenced protein complexes of their interacting proteins by mass spectrometry. This led to the identification of HuR, a mRNA stabilizing protein. This study concluded that MAT2beta variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.

  2. Lau MY, Han H, Hu J, Ji C. Association of cyclin D and estrogen receptor α36 with hepatocellular adenomas of female mice under chronic endoplasmic reticulum stress. J Gastroenterol Hepatol. 2013 Mar; 28(3):576-83.  doi: 10.1111/jgh.12084. PubMed PMID: 23216077; PubMed Central PMCID: PMC3584191.
  3. Ji C, Kaplowitz N, Lau MY, Kao E, Petrovic LM, Lee AS. Liver-specific loss of glucose-regulated protein 78 perturbs the unfolded protein response and exacerbates a spectrum of liver diseases in mice. Hepatology.  2011 Jul; 54(1):229-39. doi: 10.1002/hep.24368. PubMed PMID: 21503947; PubMed Central PMCID:  PMC3125405.

    The two publications above (Lua et al. and Ji et al.) are the direct benefits from the Subcore services. The proteomics Subcore performed a 2D-MS protein profiling for the study on organelle stress and alcohol-induced liver diseases. The proteomic results together with data from DNA microarray, real-time PCR and immunohistological analyses confirmed that perturbations of unfolded protein response (UPR) were associated with a spectrum of liver injuries in a liver specific knockout mouse model.

  4. Geary LA, Nash KA, Adisetiyo H, Liang M, Liao CP, Jeong JH, Zandi E, Roy-Burman P. CAF-secreted annexin A1 induces prostate cancer cells to gain stem cell-like features. Mol Cancer Res. 2014 Apr; 12(4):607- 21. doi: 10.1158/1541-7786.MCR-13-0469. Epub 2014 Jan 24. PMID: 24464914 [PubMed – in process]
  5. Yang H, Li TW, Zhou Y, Peng H, Liu T, Zandi E, Martínez-Chantar ML, Mato JM, Lu SC. Activation of a Novel c-Myc-miR27-Prohibitin 1 Circuitry in Cholestatic Liver Injury Inhibits GSH Synthesis in Mice. Antioxid Redox Signal. 2014 Sep 16. [Epub ahead of print] PMID: 25226451 [PubMed – as supplied by publisher].

    In this study, mass spectrometry-based proteomic identification of prohibition 1 as an ARE binding protein and its decreased binding after lithocholic acid treatment is the key finding that led to the discovery of a new molecular mechanism for chronic cholestatic liver injury involving c-Myc-miR27-prohibitin 1 regulating glutamate-cysteine ligase expression.

  6. Chang AH, Sancheti H, Garcia J, Kaplowitz N, Cadenas E and D Han. Respiratory substrates regulate Snitrosylation of mitochondrial proteins through a thiol-dependent pathway. Chem Res Toxicol. 2014 May 19;  27(5):794-804. doi: 10.1021/tx400462r. Epub 2014 Apr 23. PMID: 24716714.