Life science discoveries large and small rely on biological collections

by Kyria Boundy-Mills

Curator, Phaff Yeast Culture Collection, UC Davis

A recent high-profile scientific publication (Dash et al. 2019) caught fire in the popular press: Scientists at the University of Nebraska and Temple University found a way to eliminate HIV virus from infected mice. Their protocol may lead to a cure for HIV infection in people. Wow!! That’s big! 

Because I am a yeast collection curator, when I read scientific publications, I pay attention to what (and how many) repositories provided biological materials. I mentally give one point for each repository tapped. For example, a recent study about a bacterial plant pathogen that infests citrus used bacteria from the USDA-ARS and state repositories, insect vectors from the University of California-ANR, and citrus seeds from a program at UC Riverside. Four repositories, four points.

Reading Dash et al. 2019 was like hitting the jackpot: 6 sources in one paper! The biological materials listed in the Materials and Methods section include:

1. ·      Mice from Jackson Laboratories
2. ·      Human cell lines from ATCC and the Aids Reagent Program of NIAID
3. ·      HIV-1 virus from the NIH Aids Reagent Program
4. ·      Vectors and plasmids from Addgene
5. ·      AAV-9 virus from Vigene Biosciences Inc. (the virus that delivers the CRISPR-Cas9 treatment to the mouse genome to excise the HIV-1)
6. ·      Antibodies from BD Biosciences and BD Parmingen

MMRRC mouse line from the UC Davis Mouse Biology Program, 

photo by M. Valenzuela, provided by Renee Araiza 

But wait, there’s more! Many other items used in this study such as enzymes, CRISPR-Cas9, and antibiotics may also be linked to biological collections. The Taq polymerase enzyme used in PCR and DNA sequencing traces its origin to a strain of Thermus aquaticus obtained from the American Type Culture Collection (ATCC). When my thesis advisor Dennis Livingston was a student, he had to order specific bacteria from collections and isolate his own restriction enzymes for his experiments. Thank goodness times have changed, we can just order these materials from catalogs now. But the enzymes and other essential items we use today trace their origin to living organisms, many of which were preserved in and obtained from culture collections. 

Moral of the story: Take a moment to appreciate the many repositories that contribute to your life science discoveries.

Reference: Dash, P.K., et al., Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nature communications, 2019. 10(1): p. 1-20.

The less exciting details, for the benefit of you list-o-philes:

Materials cited in the Materials and Methods section that came from biological collections and biological supplier companies:

·       TZM-bl reporter cell line (AIDS Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD) 

·       HEK-293T cells (the American Type Culture Collection (ATCC), Manassas, VA)

·       Jurkat (Clone E6–1, TIB-152™) cells were purchased from ATCC

·       [HIV-1 Virus?] HIV-1NL4–3 

·       Vector pX601-AAVCMV: NLS-saCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (a gift from Feng Zhang via Addgene) (61591; Addgene).

·       pKLV-U6gRNA(Bbs1)-PGKpuro2ABFP (Addgene catalog #50946)

·       AAV-9 serotype (Vigene Biosciences Inc., Milton Park Abingdon, UK).

·       Mice NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were obtained from the Jackson Laboratories, Bar Harbor, ME

·       antibodies (all from BD Biosciences, San Jose, CA); Antibodies and isotype controls were obtained from BD Pharmingen, San Diego, CA,

Materials that came indirectly from biological organisms and/or collections:

·      Reagents such as ciprofloxacin, gentamicin, interleukin-2, puromycin

·      Plasmids such as pNL4–3-EGFP-P2A-Nef plasmid

·      DNA sequences used for CRISPR-Cas9; HIV-1 genome database; human genome

·      The Taq polymerase used in PCR, qPCR and Sanger and next generation DNA sequencing ultimately originated from Thermus aquaticus ATCC [number]

·      Restriction enzymes Bsa1, EcoRI, KpnI

·      Other enzymes including DNase I, proteinase K, 

·      Vectors such as AAV-CMV-saCas9 vector 

·      In-Fusion HD Cloning Kit (Clontech, Mountain View, CA)

An open letter to Jeff Bezos

June 21, 2017

An open letter to Jeff Bezos:

Living microbe collections are a foundation of the modern economy. They have contributed to healthcare, biotechnology, and studies of biodiversity, as well as animal and plant agriculture. No comparable entity has done more to promote a level playing field for economic development and yet culture collections face an existential crisis. Perhaps this is because the impact of living collections is made by the clients who obtain materials from these collections rather than by the collection itself.

According to the Convention on Biological Diversity, living microbe collections, historically called culture collections, are essential to preserve microbial biodiversity. Culture collections have been identified as a necessary development component by the Organization for Economic Cooperation and Development. With these mandates, collection groups are working to insure that they have capacity to manage the microbial resources essential for equitable sharing of the advances of biotechnology. Yet collections around the world are understaffed and spend a disproportionate amount of time working to secure funding. 

Many of the world’s leading collections, such as the American Type Culture Collection, have had to charge large fees and prioritize revenue generating materials rather than acting as a comprehensive archive.  To avoid these fees, strains are exchanged ad hoc or are isolated locally and this makes it difficult to compare or reproduce results which hinders scientific progress. Meanwhile, international initiatives such as the Global Seed Trust Svalbard Seed Vault are not incentivized to maintain microbial resources even though most plants require microbes to reach their maximum potential.

When faced with the challenge of how to use his vast fortune to benefit America, Andrew Carnegie established over 2,500 libraries at the cusp of the 20th century. Now as we face the dawn of the biotechnology era at the beginning of the 21^st century, the time is right for a philanthropist to establish a network of open, public living microbe collections.

These collections will preserve and archive living microbes used in research and technology and insure that the materials are openly available to qualified recipients. They will advance the rise of biotechnology by insuring that high quality materials are used and that the exact same materials are used by different investigators. Much as the printing press allowed books to be copied exactly, living microbe collections can provide the exact same strain to multiple clients, and still have the original.

You asked for ideas that would change the world; living microbe collections are exactly the sort of investment that could change the world. Additionally, Living microbe collections are poised to benefit from more than just money. They are uniquely positioned to take advantage of the Amazon database, e-commerce, fulfilment, and cloud computing resources and with the appropriate investment are ready to be the rising tide that lifts all boats.

Mr. Bezos, secure your legacy by supporting an international network of living microbe collections that engage and benefit from the Amazon resources and infrastructure.

Humbly yours,

Kevin McCluskey
Curator, Fungal Genetics Stock Center
Research Professor
Department of Plant Pathology
Kansas State University
785-532-1420
mccluskeyk@ksu.edu

@theFGSC
www.fgsc.net Fungal Genetics Stock Center
www.usccn.org US Culture Collection Network
www.wfcc.info World Federation for Culture Collections

The following letter was written by living collection curators and sent to the NSF on June 2, 2016



June 2nd, 2016

James Olds, PhD Assistant Director for Biological Sciences         National Science Foundation 4201 Wilson Boulevard Arlington, VA 22230

Muriel Poston, PhD, JD Director of the Division of Biological Infrastructure National Science Foundation 4201 Wilson Boulevard Arlington, CA 22230

 

Dear Drs. Olds and Poston,

 

The living collections community appreciates the opportunity to provide feedback to the Collections in Support of Biological Research (CSBR) program within the Directorate for Biological Sciences at the National Science Foundation. The living collections community includes dozens of scientists that serve as the directors, managers, and curators of over 20 living collection resources that are invaluable long-term infrastructure investments. We also hope to represent the thousands of scientists who utilize our living stock centers as fundamental resources for their individual research programs, many of which are supported by NSF research grants. As a community, we appreciate this opportunity to express our concern for the 2016 CSBR funding hiatus, hoping to provide constructive feedback that represents collective input from the US living collections community and our constituents. We have been pleased to learn that the NSF has announced a return to accepting proposals for CSBR in 2017, though we nonetheless have concerns about the adequacy and reliability of resource support going forward. The living stock collections that we oversee span the Tree of Life (from bacteria to plants, fungi, and animals), and provide the foundational tools necessary for research in nearly every biological discipline including basic and applied research with spin-off impact in diverse areas of technology and engineering at every scale. These collections are the result of decades of careful acquisition, curation, quality control, and stewardship. They are each unique and irreplaceable. Moreover, they serve as a resource for the education community and are heavily used by educators from K12 through the university level. Contributions to education come in the form of elementary school science fair projects, citizen science ventures, high school science labs, university science curricula, and training grounds for the next generation of STEM scientists. Finally, they encourage strong community engagement through contributions to museums, zoos and other institutions of public interest.

1. Scope of Collection Support

The living collections community has multiple concerns with regard to the adequacy and scope of collection support provided by CSBR, enumerated here, and further amplified below: 1) The inconsistent funding environment with large program changes over the last six years has been disruptive, and consequently, has curtailed efforts towards improving best practices and curatorial innovation, 2) Careful financial analysis has repeatedly shown that the expectation for living collections to be self-supporting and sustainable is unrealistic, and efforts on our part to increase user fees have created a sense of distrust and insecurity amongst our user base, and 3) By combining the diverse and sometimes conflicting needs of living and natural history collections into a single resource pool, CSBR has inadvertently underserved both communities and created a false sense of competing agendas. Rather, both communities should be encouraged to cooperate and send a unified message to the governing authorities who control federal research dollars. The inconsistent support for living collections by NSF has resulted in a variety of negative outcomes. First, it sends a message to hosting institutions, supplemental funding sources, and to the research community that living collections are low priority infrastructure resources. This implicit message results in reduced institutional support and increased difficulty in acquiring support from additional funding sources. Inconsistent support for infrastructure resources also has the potential to negatively impact the quality of stocks maintained by compromising salary continuity, and leads to gaps in the implementation of quality control protocols to verify stock health and identity.
The living collections community is also concerned about recent initiatives by NSF that living collections must progress to self-sufficiency. This expectation is unrealistic for two fundamental reasons: 1) a self-sufficiency model shifts all costs to the end users who themselves are struggling with limited research dollars, and 2) this model sharply reduces institutional incentives to host living collection resources. In our collective experience, when costs are shifted to the end-user, the increase in fees drives down stock utilization by as much as 50%, which results in further financial challenges for the collection. Reducing access to strains has a larger impact on smaller labs, institutions, and training programs relative to larger labs with more resources. Equal access to stock repositories by researchers should be considered a priority of funding agencies. The expectation that living collections be self-supporting also restricts research endeavors to a small number of research systems with sufficient internal resources and creates obstacles to progress rather than enabling diverse and innovative research activities. In the worst cases it will lead to collection closure, but in all cases it will force collections to decide between quality of the collection and the diversity that the collection holds. Similarly, when stocks are not available from central repositories, there is an increase in ad hoc exchanges, often without regard to regulations on movement of genetically modified or potentially pathogenic or invasive organisms.

2. Proposed Solutions for Living Collections Support

Our fundamental recommendation is that the NSF return to a separate panel model wherein living stock centers are considered and funded independently from fossil and other archival natural history collections. While the overarching aims for the documentation and study of life on earth are shared by the living stocks and museum resource centers, they differ in fundamental ways that should be individually treated by the NSF and by the research community. Whereas archival collections are fundamental to understanding the past, living stocks centers are critical for examining biological phenomena in real time. Real-time analysis of organismal "behaviors" spans scientific disciplines as diverse as molecular evolution via analysis of genomic mutation rates, to disease transmission, reproductive biology, cancer genetics, behavioral ecology, cognition, cell biology ... the list is truly limitless. One exceptional example is the ability to use CRISPR/Cas9 gene editing tools on stocks from living collections. This uniquely situates living stock collections to provide resources that link research through time as new technological advances are added to our toolkit. The dynamic nature of living stock collections allows them to parallel advances in research by enabling incorporation of novel stock types as the community develops them.
The current mechanism for evaluating living stocks and archival natural history collections within a single panel has inadvertently undervalued the value and the objectives of both categories of resource center. In addition to the differences outlined above, living collection staff are not typically funded by the host institution because they serve the research community at large. Thus, unlike the case with natural history collections, interruptions in funding will assuredly lead to immediate loss of personnel, loss of stocks, and even loss of whole collections.
The living collections community therefore respectfully requests increased funding stability, with a transparent directive from NSF, which will support competitive living collection proposals at a level covering at least 40% of the operating budget. Criteria for competitive proposals should include maximizing efficiency with a reasonable budget based on the number of stocks maintained, types of ‘products’ and support services provided, diversity of use, and distribution. One suggestion for improving stability is to explore new funding models with cross-agency (e.g. USDA, NIH, DOE) funding opportunities. New funding models should be explored in a joint meeting with members of the living collections community, funding agency representatives, and facilitated by FASEB, NSCA, APS or other wide-reaching societies.

3. CSBR Program Impact

Within the category of impacts of the CSBR program, the living collections community is particularly concerned about the expectation for collections to conduct innovative research in addition to infrastructure maintenance. The living collections community feels that the present three year project-based grants are a good mechanism to promote innovation and outreach but should be separated from sustenance support which can highlight efficient operation, quality management, and community engagement instead. The purpose of infrastructure is to provide access to high quality, professionally managed resources. One important evaluation metric, described below, is the number and type of publications depending on living collection resources.
As with all NSF-funded programs, there has been increased expectation for education and outreach programs. We embrace this mandate, and when sustenance support is provided on a long-term basis, outreach can extend beyond the individual research community supported by each living collection. Student engagement, safe and validated resource provision, and opportunities for educator involvement in long-term projects are facilitated. Thus, reliable, transparent, and quantitative mechanisms for collections support not only enhances scientific output and merit, it also powerfully enables the living collections community to perform its educational functions.

4. Evaluation Metrics

While it is difficult to generate a life-long tally, living collections resources have been cited in tens of thousands of publications, have generated a multitude of patents, and have been at the forefront of innovative technologies for decades. The pseudo h-index metric, which shows the number of highly cited publications, is being used at many collections and demonstrates impacts substantially higher than those produced by individual scientists.
The efficiency of maintaining living stocks in central repositories is decidedly superior to the alternative of maintaining duplicate lines. Thus, the most important metric for measuring the impact of a living collection is the total number of stocks provided to the community, and the diversity of institutions to which they are provided. There are numerous instances wherein multiple labs have created strains that have been misidentified through ad hoc maintenance. Support for central repositories for widely used stocks eliminates the need for individual labs to waste time and money collecting the organisms, creating new lines, maintaining duplicate research material, and distributing stocks to the community --- but most importantly, professionally managed stock centers assure quality control and scientific repeatability.
Living collections are repositories that provide an avenue for repeatability that could not be substituted by any other mechanism. Repeatability is central to all scientific endeavors as was recently highlighted in Science. For many experiments, it is critical that precisely the same isolate or strain be used to ensure that the results are comparable. The utilization of the same stocks for a wide range of comparative experiments would be impossible without living collections, as some of the most widely utilized (and effective) stocks have been maintained successfully for more than 25 years, and include several model organisms, including biodiversity and type specimens.
Several federal agencies and societies are encouraging initiatives for journals to require that manuscripts document and acknowledge use of infrastructure resources, including stock numbers from living collections. This type of data is only available from living collections that maintain detailed records of their stocks, and provide details for the specific colonies/species used.

5. Call for Action

Living collections are the product of decades of careful curation, development of unique expertise, and collection and storage of associated metadata. Living collections staff are uniquely knowledgeable, and via interaction with the research community, provide irreplaceable expertise on choosing appropriate stocks, executing experimental design, and maintaining appropriate and sustainable husbandry practices. This level of experience leads to efficiency from the perspective of the collection, but also from the perspective of the researcher using the collection.
Most stocks in living collections have been accumulated over decades and are irreplaceable. Permits to collect in the wild are increasingly difficult to obtain, and/or funding is not available for collecting trips. Current living stocks have been generated through the work of large numbers of individuals --- often funded through NSF grants --- over the course of many years. Thus, while it is nearly impossible to put a monetary value on biological collections, it is manifestly clear that ad hoc management of living stock collections would result in loss of scientific repeatability, breaches in regulatory compliance, and untenable risks associated with independent professional collections. We urge the NSF to consider the unique value of established living collections, the scientific rigor and promise that they represent, and the impossibility of recreating these resources once they are lost by reinvesting in their continued maintenance and growth.

Respectfully submitted by:

Dr. Jelena Brkljacic

Associate Director, Arabidopsis Biological Resource Center
(Ohio State University)

Dr. Michael Felder

Director, Peromyscus Genetic Stock Center
(2005-2015) (University of South Carolina)

Dr. Erich Grotewold

Director, Arabidopsis Biological Resource Center
(Ohio State University)

Dr. Hippokratis Kiaris

Director, Peromyscus Genetic Stock Center
(University of South Carolina)

Dr. Pete Lefebvre

Co-director, Chlamydomonas Resource
Center (University of Minnesota)

Dr. Michael W. Lomas

Director, Provasoli-Guillard National Center for Marine Algae and Microbiota
(Bigelow Laboratory for Ocean Sciences)

Dr. Therese Markow

Director, Drosophila Species Stock Center
(2000-2016) (University of California San Diego)

Dr. Kevin McCluskey

Curator, Fungal Genetics Stock Center
(Kansas State University)
World Federation for Culture Collections Executive Board

Dr. David Nobles

Curator, Culture Collection of Algae
(University of Texas Austin)

Dr. Patrick O’Grady

Director, Drosophila Species Stock Center
(2017) (University of California Berkeley)

Dr. Maxi Richmond

Assistant Director, Drosophila Species Stock Center
(University of California San Diego)

Dr. John Wertz

Director, E. coli Genetic Stock Center
(Yale University)

Dr. Anne Yoder

Director, Duke Lemur Center
(Duke University)

Dr. Daniel R. Zeigler

Director, Bacillus Genetic Stock Center
(Ohio State University)

NSF CSBR Funding Hiatus- A letter from the living collection community

April 26, 2016

Dr. James L. Olds
Assistant Director
Directorate for Biological Sciences
US National Science Foundation
4201 Wilson Boulevard
Arlington, VA 22230

Dear Dr. Olds,

The US Culture Collection Network and its stakeholders appreciate the opportunity to comment on the hiatus in accepting proposals by the US National Science Foundation Collections in Support of Biological Research (CSBR) program. If a picture is worth a thousand words, the value of the physical object itself is even higher. Biological specimen collections including microbial culture collections strive to organize, preserve, characterize, and distribute the actual objects described in scientific research.

Formal, organized biological collections are an essential component of research infrastructure. As a special category of biological collection, living research collections allow generations of scientists to work on professionally validated and preserved living materials, minimizing specimen loss, genetic drift and contamination. These types of collections are undergoing expansion in the volumes and types of data connected to the specimens, the intrinsic value of the materials in economic and regulatory terms, and in the public awareness of the need for core support to these resources. Living collections also foster reproducibility of scientific experiments, in an increasing diversity of research areas.

An enormous range of life science research at academic institutions, government agencies, and companies depends on the three interdependent resources offered by living stock collections: the specimens, the associated data, and the expertise of the collection curators. Organisms are being used in ways the scientists who deposited them into collections decades ago never could have imagined, such as development of PCR based on discovery of heat-stable DNA polymerase produced by an obscure bacteria preserved in the American Type Culture Collection (ATCC) for decades.

Thus, living collections are the libraries of biology. Thanks to advances in biochemical genetics and whole genome sequencing, we are able to “check out” samples from these libraries, knowing its exact characteristics from prior work – in many cases from studies funded in past decades by NSF, and/or involving database programs funded by NSF. These characteristics include things like antibiotic or anti-cancer drug production, the ability to benefit or harm agriculture, the use in food or fiber production and processing, in biofuel production, and as reagents for diagnostic and environmental tests to keep our world safe.

Through a 2012 Research Coordination Network grant by the NSF CSBR program, a community of living collection scientists called the US Culture Collection Network have made significant progress in implementing best practices, developing shared resources, speaking with a common voice, and in seeking alternate support. Because the materials in living collections cannot survive without curation, gaps in support endanger the very existence of these collections. Unlike natural history collections, living organisms that require tending cannot physically survive a “hiatus”; the stocks die.  Similarly, funding gaps and insecurity create staffing challenges.  Because of their inherent long-term nature, collections require staff with specialized skills and knowledge, including knowledge of the collection history and holdings, taxonomy, database and website management, quality management, shipping regulations, biosafety regulations and international treaty obligations pertaining to genetic resources.

Many of the living microbe collections in the US that are particularly valuable because they contain material from different places, times, or branches of the tree of life, are endangered or orphaned due to retirement, decreased funding, or increased regulatory restrictions. Many of the isolates in these endangered living microbe collections will be impossible to replace due to habitat loss and changes in genetic resource ownership.  The NSF CSBR program has been the primary extramural funding source to rescue these irreplaceable collections, but is already insufficiently funded to fulfill the existing need. 

Living collections, including microbe, plant, and  animal collections, make every dollar in federal biological science research funding go farther. They allow scientists at different institutions, and even from different eras, to work on the exact same living materials. Simply put, living collections ensure “apples to apples” comparisons. When living research materials are available at low cost and from authoritative sources, regulatory compliance and public safety are enhanced.  By way of contrast, when the full cost of maintaining these resources is borne by the end-user, the ability to conduct pilot studies to lay the foundation for grant proposals, the ability of researchers at historically under-served institutions to engage in modern research, and by extension, the use of well-qualified biological materials in secondary, post-secondary, and graduate training is compromised. Successful models have demonstrated that institutional and governmental funding of biological collections assures a more even playing field, providing affordable access to fundamental research materials.

For many decades, the US National Science Foundation has been a leader in supporting diverse living collections at universities and non-governmental institutions. Among these, collections of algae, bacteria, fungi, and yeasts support diverse research communities and complement, but do not overlap significantly with other public collection holdings.  The USDA microbe collections are managed as an intramural program, and the US NIH disbanded the National Center for Research Resources under P.L. 112-74 in 2011. Some medically oriented microbe collections are operated under contract by the ATCC and some specialized collections receive support from different institutes at the NIH, creating an uneven playing field. Moreover, because the ATCC has had to be self-supporting for several decades they have charted an independent path that precludes managing the large numbers of microbial isolates used in active research.

The hiatus in accepting grant proposals for Collections in Support of Biological Research at the US National Science Foundation impacts not only the collections themselves, but the thousands of users who depend on these collections. The Natural Science Collections Alliance, Society for the Preservation of Natural History Collections and the American Institute of Biological Sciences recently expressed their desire for a return to funding collections and the American Phytopathological Society has long been a leader in advocating for living microbe collections. In signing this letter, we add our voices to this effort to encourage not just a cancellation of the hiatus, but an increase in support for living scientific collections for research and education.

Thank you for considering this letter in support of funding living collections as a fundamental component of a mature science infrastructure. 

Kevin McCluskey, PhD

Curator, Fungal Genetics Stock Center
Kansas State University

Kyria Boundy-Mills, PhD
Curator, Phaff Yeast Culture Collection
University of California, Davis

David Nobles, PhD
Curator, UTEX Culture Collection of Algae

University of Texas, Austin

John E. Wertz, PhD
Director
E. coli Genetic Stock Center
Yale University

David Smith, PhD
Director of Biological Resources
CAB International

Surrey, UK

Seogchan Kang, PhD
Professor, Department of Plant Pathology & Environmental Microbiology
Penn State University
University Park, PA

Jessie A. Glaeser, PhD
Team Leader, Center for Forest Mycology Research
US Forest Service, Northern Research Station
Madison, WI