A Sandia team led by researcher Anup Singh (8321) is taking a new approach to studying how immune cells respond to pathogens in the first few minutes and hours of exposure.
Their method looks at cells one at a time as they start trying to fight the invading pathogens.
Called the Microscale Immune Studies Laboratory (MISL) Grand Challenge, the work is in its second of three years of funding by the internal Laboratory Directed Research and Development (LDRD) program. Sandia is partnering on the project with the University of Texas Medical Branch (UTMB) at Galveston and the University of California, San Francisco (UCSF).
Anup says the researchers are interested in studying the early events in immune response when a pathogen invades a body. Understanding the early steps could lead to better ways to diagnose and stop disease before there are symptoms, and development of more effective therapeutics.
Most existing research into how immune cells respond has been done by looking at large cell populations. The Sandia researchers say information gathered from a large population of cells may mask underlying mechanisms at the individual cell level.
“Cells have different life cycles, just like any living being. And not all cells are exposed to the pathogen at the same time,” Anup says. “We wanted to look at cells in the same life cycle and same infectious state. This can only be done cell by cell. We also want to study populations, but one cell at a time.”
The research is possible because of advances in several Sandia-developed tools, including:
“Early on we realized that we did not have sufficient biological expertise needed for this project, so in addition to building collaborations with universities, we aggressively pursued hiring biologists at Sandia,” Anup says. “The addition of five new biologists has greatly increased our ability to develop biological understanding and reagents required to perform MISL experiments.”
Real immune cells are short-lived outside of bodies. To do the type of experiments they wanted, the researchers needed cells that can stay alive more than a couple of hours, have the ability grow, and represent a relevant model of human immune cells. They obtained “immortalized mouse immune cells” from a collaborator at UCSF that have the needed life span, and are accepted as a model system by the immunology research community.
“We’re starting with robust and well-characterized cells, which really simplifies development of our new technologies and methods, says biologist Steve Branda (8321). “We’ll soon be working with other cell types, though, like white blood cells directly isolated from human patients. Our approach is designed to be flexible enough to handle many different cell types, and it also minimizes the number of cells needed for analysis, so it should enable us to do some unique studies on rare cell types.”
Proteins in the cells of interest are tagged with fluorescent molecules, essentially colored dyes. The dyes range from green to red and give researchers the opportunity to track proteins and see, for example, the dynamic cellular production of proteins or protein-binding processes inside or on the surface of the cells.
The team is developing one platform with two complementary microfluidic modules — one developed for trapping and imaging viable cells during stimulation with pathogens. The second module combines cell preparation steps, cell selection, and sorting followed by analysis of protein content in the selected cell subpopulations.
Mechanical engineer Amy Herr (8321) and coworkers are working on the module that allows for sensitive, robust, and rapid protein quantification. They are analyzing protein levels and protein modifications in both single-cell and small cell populations of less than 1,000 cells at critical time points in the pathogen invasion. The engineering team interfaces directly with Sandia biologists, allowing the engineers to both develop methods useful to addressing biological hypotheses and validate the new tools against accepted methods.
“Specifically our module seeks to quantify protein events with sensitivity that is not currently attainable,” Amy says. “Further, we have designed tools that allow complete control over cell introduction, challenge, and analysis — thus enabling measurements of special interest to the ongoing predictive simulations.”
Conrad James (1744) and his team are working on the module for trapping and arraying cells so they can be imaged, and ensuring that cells are kept alive and healthy during the experiment. Hyperspectral fluorescence imaging with multivariate curve resolution (MCR) is then used to provide quantitative measurements on multiple proteins simultaneously. The goal is to analyze as many as 10 to 40 proteins and cellular stains at a time in three dimensions.
David Haaland (8332), lead member of the hyperspectral fluorescence imaging team, says his group provides 3D hyperspectral fluorescence imaging of 15-micrometer-diameter cells and their interactions between the cells in real time.
“This gives us the unique ability to quantitatively image many labeled molecules simultaneously in the cells during the host-pathogen interactions,” he says.
The end results of the imaging and protein analysis are large amounts of data that must be categorized and understood. That’s where computation modeling comes into play, says Jean-Loup Faulon (8333), coordinator of the computational core of the project.
“The goal of the computational core is twofold — to generate hypotheses to be measured experimentally by the biology and platform cores, and to produce a predictive model of immune responses,” he says.
Hypotheses are generated using a variety of bioinformatics tools to predict novel interactions between proteins and regulators involved in the innate immune pathways. The predictive model makes use of stochastic dynamics simulations — processes that can be described by a probability distribution. These can be used to ask and answer “what-if” questions about cell pathway responses and complement the experimental efforts.
The computational modeling is performed at both Sandia/California and Sandia/New Mexico.
Anup says using an integrated microfluidic platform sets Sandia apart from the rest of the world. Sandia researchers have been working in the area of microfluidics — the science of designing, manufacturing, and formulating devices and processes that deal with volumes of fluid on the order of nanoliters — since the 1990s and have a good understanding about how to use microfluids to analyze cell activity. The microfluidic platform is fast and highly parallel and can perform hundreds of measurements 50 to 100 times faster than alternate methods.
Sandia’s growing cadre of biological scientists provides key contributions to the grand challenge through the biology core of the project, coordinated by Tony Martino (8332).
“This project integrates a number of areas in which Sandia has a lot of expertise,” Tony says. “We are bringing together host-pathogen biology, cell and protein manipulation using microfluidic and BioMEMS technologies, and computational biology. We are building something bigger than the sum of the parts, and that is a great strength for Sandia.”
He adds, “We are challenging the way people think about doing biological experimentation. Single-cell measurements and simultaneously measuring protein behavior when there might be just one or a few molecules present will revolutionize biology.”
Anup says the end goal is to make a benchtop miniaturized system expected in about two years. It would be placed in Biosafety Level 3 or 4 labs to study immune response to highly pathogenic organisms. Moreover, the integrated platform, biological reagents and computational models developed under this project have applicability beyond infectious disease research. These technologies can also be used for studying cellular signaling involved in diseases such as cancer or by pharmaceutical companies for biomarker discovery.
Glenn Kubiak (8320), MISL project manager, emphasizes the importance to the project of the partnerships with UTMB and UCSF.
“Sandia’s expertise in microsystems, advanced chemical imaging, and computing, combined with their expertise in emerging infectious disease and cellular signaling has created a team that is unique in its ability to contribute both to defense against infectious diseases and to therapeutics,” he says. “Folks we’ve briefed in government agencies and companies have been pretty amazed by the strength of our partnership and also by the audacity of what we’re doing together. That’s why we call it a grand challenge.
Principal investigator: Anup Singh (8321)
Project manager: Glenn Kubiak (8320)
Platform core: Anup Singh (8321, coordinator), Amy Herr (8321), Igal Brener (1721), Jim Brennan (8321), Susan Brozik (1714), Conrad James (1744), Ron Manginell (1744), Matt Moorman (1744), Kamlesh Patel (8324), Thomas Perroud (8324), Surendra Ravula (1727), Ron Renzi (8125), Nimisha Srivastava (8321), Dan Throckmorton (8321)
Biology core: Tony Martino (8332, coordinator), Steve Branda, Catherine Branda, Zhaoduo Zhang, Todd Lane, Meiye Wu (all 8321), Jens Poschet (8332), Bryan Carson, Roberto Rebeil , Bryce Ricken, Kevin Crown, Amanda Carroll-Portillo (all 8331),
Imaging team: David Haaland (8322), Mike Sinclair (1824), Howland Jones (8332), Mark Van Benthem (8332), Rachel Noek (8332), David Melgaard (5534), Chris Stork (1823)
Computational core: Jean-Loup Faulon (8333, coordinator), Jaewook Joo (8333), Shawn Martin (1412), Steve Plimpton (1412), Susan Rempe (8333), Ken Sale (8321) -- Chris Burroughs
By Patti Koning
School children in Livermore have seen a ring nebula up close. They’ve traveled across our solar system to Pluto and back again. They’ve visited other galaxies and seen the sun up close. All thanks to Sandia.
The Labs provided crucial funding in purchasing a Digitalis portable planetarium for the Livermore school district. The funding came through Lockheed Martin’s Gifts and Grants program, which Sandia/California distributes to organizations in the community.
“Bringing this incredible teaching tool into the local schools can hardly be more wonderful,” says Sandia Community Relations Officer Jim Simmons (8528). “There is a growing national concern about science education, which we can use our resources to address. This is a perfect example of what we want to accomplish in community relations.”
The planetarium is a simple device, consisting of a 10-by-16-foot dome tent, a projector, and a computer. Stellarium, an open-source planetarium program, provides the magic — the images of the stars, planets, and anything else you can find in space.
At a dedication ceremony for the planetarium, Livermore schools Superintendent Brenda Miller said, “This is what true education is — when you can bring a tool into the schools and generate so much excitement.”
Sandia not only provided the bulk of the funding for the planetarium, but also the brainpower to prepare the tool for use in the schools. The driver behind the planetarium effort was the Teaching Opportunities for Partners in Science (TOPS) program, which places retired scientists and engineers in the classroom.
The Livermore TOPS program includes retired engineers and scientists from Sandia, Lawrence Livermore National Laboratory, General Electric, and other local technology companies.
When several TOPS scientists borrowed a planetarium from the San Joaquin County Office of Education last year, they decided they had to have it for the Livermore schools. Cost was an issue, as the price tag for the planetarium is $25,000, plus operation and maintenance costs. Still, the TOPS scientists were well on their way with two donations of $5,000 each from General Electric and community member Prabha Duneja.
Jeff Manchester, manager of Public Relations and Strategic Communications Dept. 8528, recalls being asked if Sandia would kick in another $5,000.
“We decided we’d rather just solve the problem, and we donated $20,000 to complete the purchase,” he says. “The planetarium was a wonderful opportunity to provide a science-directed solution for elementary and middle school students.”
Sandia retiree Ken Mitchell was part of a team that spent several weeks in December preparing the planetarium for use in schools. The work included writing a user manual, testing the system, and developing scripts to guide teachers through lessons on the moon, distant galaxies, constellations, and planets.
Ken, who spent more than 40 years at Sandia working in stockpile engineering, has been working at Marylin Avenue Elementary School as a TOPS scientists since he retired in 2000. In January, he led lessons in the planetarium at Marylin.
“The students just love it. They say it’s the best science day they’ve ever had,” he says. “Most kids never even look at the sky at night, but after a lesson in the planetarium they can recognize the Big Dipper, Orion, and other constellations.”
When conducting lessons in the planetarium, Ken prefers to stray from the scripts for a more interactive experience. He’ll ask the kids questions about what they already know and use that as a basis for delving deeper.
Ken thinks they’ve just touched the surface of the planetarium’s potential as a teaching tool. In addition to astronomy, there are also social studies lessons to be learned. The constellation script not only shows the major constellations, but also demonstrates how the Egyptian, Roman, Chinese, Hindu, Inuit, and Navajo cultures interpreted the stars.
Ken and other TOPS scientists are now working to develop more lessons based on the state content standards and applications for middle school students. In fact, the biggest limitation of the planetarium is time. With 10 elementary schools and four middle schools in Livermore clamoring for it, the planetarium has to move quickly between sites to give all students an opportunity. -- Patti Koning
By Neal Singer
A Sandia physicist remembers all too vividly how difficult the transition from postdoc to technical staff at Sandia can be. So she has helped start an organization to enable current postdocs to network, learn about research going on outside their own cubbyholed areas, and develop professional techniques to help their careers blossom.
“There’s no reason that the career skills I learned about at the last minute should not be available to appointees throughout their postdoc experience,” says Gayle Thayer (5711).
At the first meeting last Tuesday of the PostDoctoral Professional Development Program (PD)2P, the principal speaker was, fittingly enough, 1000 VP Rick Stulen, who had hired his first postdoc in the early ’80s before the practice became formal policy at Sandia.
Rick, speaking to about 75 postdocs and mentors in Albuquerque and about 50 videoconferenced at Livermore, says, “I saw the impact [of postdocs hired] at Lawrence Livermore National Labs and latched on to the concept. The level of work was good, and the enthusiasm was personally enriching.”
The expectations of a Sandia postdoc, he says, should be “to have a solid mentor who is well-connected to what you care about technically, who connects you to other parts of the lab, who cares about your career goals, and who helps access facilities you can’t find elsewhere. You’re losing a little bit of the Labs experience if you don’t take advantage of those facilities.”
An open question remained as to whether Sandia should follow the recent leads of its sister labs, Los Alamos and Lawrence Livermore, by formalizing the relationship between postdocs and the Laboratories, with courses for advisors on how to mentor, opportunities for learning and advancement provided to mentees, and more comprehensive definitions of the terms of engagement. Other issues raised included whether postdocs should fill out PMFs (Performance Management Forms) and be able to participate in the LDRD (Laboratory Directed Research and Development) office’s call for proposals.
Peter Feibelman (1130) said that “because Sandia has no formalized program, the work has been ad hoc, with responsibilities not clear. At LANL, they have an organization charged with improving the experience of postdocs. It’s nice that some people [here] have decided to start an organization like this, but really it’s the Labs’ responsibility.”
The Lab News asked Rick why Sandia seemed to be following other labs in this effort rather than leading.
“First,” he said, “both LLNL and LANL are run by a university. They tend to think more about education. We’re a corporate-run engineering lab whose existence is based on deliverables. It’s a different outlook. For LANL, providing a good experience to postdocs is a method of keeping up the flow of new hires from that group.
“In my view,” he said, “a healthy postdoc program is critical to Sandia. It keeps us intellectually healthy and challenged. There’s an injection of new ideas that contribute to laboratory vitality. We have a responsibility to the nation to be part of this channel. And students from industry and universities help us form a network we can rely on in our later work.”
The new organization may help further these ends.
“Currently, there’s no easy pathway to find out what’s going on in a large laboratory,” said postdoc Justin Serrano (1513). “If you don’t have a way to talk to other people, you’d never know what’s happening beyond your building or center.” The Lab News, he said, shows only high-profile work that helps him less than personal communication would.
The (PD)2P mission statement — “to become a preeminent postdoctoral springboard for the scientific leaders of tomorrow” — and its goal — “to facilitate postdocs transitioning into careers as outstanding independent researchers by providing resources for professional development, and to formalize a visible program to organize and network postdocs and highlight postdoc work” — were backed by a calendar listing career development workshops for postdocs and technical seminars by postdocs, as well as the stated intent to track postdocs after they leave Sandia to see where they went and keep the names available for future collaborations and networking.
Workshops included “How to package yourself for jobs in industry,” to be led by Rochelle Lari; “The successful postdoc,” by Peter Feibelman; and, later, how to obtain academic positions, mentorship, and grant writing.
More information can be found at the group’s website, www.sandia.gov/pd2p.
Current team members include Linda Canaan (8524), Erica Corral (1815), Anna Gorman (1815), Lisa Gray (8350), Bernadette Hernandez-Sanchez (1815), Tom Hinklin (1815), Wontae Hwang (8362), Sebastian Kaiser (8351), Gayle Thayer (5711), and Dominique Foley Wilson (1012).
The (PD)2P is supported by Wendy Cieslak (1010) in the Science, Technology, and Engineering Strategic Management Unit’s Strategic Initiatives Office, with funding from the Nuclear Weapons (NW) People Readiness Portfolio that nurtures NW-critical capabilities for the future of Sandia. -- Neal Singer