Strategies for Shared Research Resources for Enhancing Research Sustainability: Communicating Between Stakeholders and Managing Expectations to Maximize Value and Impact.

Shared research resources occupy a unique role in the scientific research landscape. Sometimes called core facilities, shared research resources provide instrumentation, services, and expertise to a wide range of researchers. With dedicated staff maintaining instruments, training users, and supporting collaborations, these resources are well situated to churn out reproducible high-quality data, lead research innovation, create efficiencies, and stimulate economic development all while driving down capital costs for institutions. That being said, in the high-paced disciplines of science with limited resources and competing priorities, these resources are often obligated to demonstrate their worth, especially beyond traditional service delivery models. How can shared research resources quantify and communicate their value and impact to stakeholders for optimal support and sustainability? For best approaches towards value proposition, it is important to understand the various stakeholders in the shared research resource ecosystem, including their needs, expectations, and value systems. This will in turn inform models of support and best approaches for planning, positioning, managing, evaluating, and improving shared research resource output to return the most value to all stakeholders involved. It is imperative that communication is tailored for each unique group of stakeholders, and terminology and expectations are managed accordingly. This work attempts to curate and share approaches and best practices toward this effort, gathered through available literature and focused engagement with various shared research resource stakeholders.

A Shift in Our Thinking: Reframing Shared Research Resources as Investments in Education and Innovation, Not Subsidized Science.

For many researchers, Shared Research Resources are often the most cost-effective means of using state-of-the-art (not to mention expensive) instrumentation. Along with access to the instruments themselves, Shared Research Resources also offer individualized training by highly qualified Shared Research Resource staff-again at deeply discounted costs compared to the operational costs of the facilities. Traditionally, this gap in revenue has been termed a subsidy. But, as with many words, connotation matters, and we posit that this language ought to be changed to reframe our thinking and impart the true impact of Shared Research Resources. We argue here that rather than a subsidy, the revenue gap is better described as an investment. Furthermore, investments of Shared Research Resources lead to positive externalities, including education and innovation.

Establishing a national strategy for shared research resources in biomedical sciences.

Contemporary science has become increasingly multi-disciplinary and team-based, resulting in unprecedented growth in biomedical innovation and technology over the last several decades. Collaborative research efforts have enabled investigators to respond to the demands of an increasingly complex 21st century landscape, including pressing scientific challenges such as the COVID-19 pandemic. A major contributing factor to the success of team science is the mobilization of core facilities and shared research resources (SRRs), the scientific instrumentation and expertise that exist within research organizations that enable widespread access to advanced technologies for trainees, faculty, and staff. For over 40 years, SRRs have played a key role in accelerating biomedical research discoveries, yet a national strategy that addresses how to leverage these resources to enhance team science and achieve shared scientific goals is noticeably absent. We believe a national strategy for biomedical SRRs-led by the National Institutes of Health-is crucial to advance key national initiatives, enable long-term research efficiency, and provide a solid foundation for the next generation of scientists.

Fully reduced granulin-B is intrinsically disordered and displays concentration-dependent dynamics.

Granulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (Pgrn). All seven Grns (A-G) contain 12 conserved cysteines that form 6 intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-functional roles, including wound healing, embryonic growth, and inflammation and are implicated in neurodegenerative diseases. Despite their manifold functions, there exists a dearth of information regarding their structure-function relationship. Here, we sought to establish the role of disulfide bonds in promoting structure by investigating the fully reduced GrnB (rGrnB). We report that monomeric rGrnB is an intrinsically disordered protein (IDP) at low concentrations. rGrnB undergoes dimerization at higher concentrations to form a fuzzy complex without a net gain in the structure-a behavior increasingly identified as a hallmark of some IDPs. Interestingly, we show that rGrnB is also able to activate NF-κB in human neuroblastoma cells in a concentration-dependent manner. This activation correlates with the observed monomer-dimer dynamics. Collectively, the presented data establish that the intrinsic disorder of rGrnB governs conformational dynamics within the reduced form of the protein, and suggest that the overall structure of Grns could be entirely dictated by disulfide bonds.

Purification and biochemical characterization of Brazil nut (Bertholletia excelsa L.) seed storage proteins.

Brazil nut storage proteins, 2S albumin, 7S vicilin, and an 11S legumin, were purified using column chromatography. Analytical ultracentrifugation of the purified albumin, vicilin, and legumin proteins, respectively, registered sedimentation coefficients of 1.8, 7.1, and 11.8 S. Under reducing conditions, the major polypeptide bands in 2S albumin were observed at 6.4, 10-11, and 15.2 kDa. The 7S globulin was composed of one 12.6 kDa, two approximately 38-42 kDa, and two approximately 54-57 kDa polypeptides, whereas the 11S globulin contained two major classes of polypeptides: approximately 30-32 and approximately 20-21 kDa. The 7S globulin stained positive when reacted with Schiff reagent, indicating that it is a glycoprotein. The estimated molecular mass and Stokes radius for 2S albumin and 7S and 11S globulins were 19.2 kDa and 20.1 A, 114.8 kDa and 41.1 A, and 289.4 kDa and 56.6 A, respectively. Circular dichroism spectroscopic analysis indicated the secondary structure of the three proteins to be mainly beta-sheets and turns. Emission fluorescence spectra of the native proteins registered a lambda(max) at 337, 345, and 328 nm for 2S albumin and 7S and 11S globulins, respectively. When probed with anti-Brazil nut seed protein rabbit polyclonal antibodies, 7S globulin exhibited higher immunoreactivity than 2S albumin and 11S globulin.

Binding of europium(III) ions to RNA stem loops: role of the primary hydration sphere in complex formation.

Understanding the process by which RNA molecules fold into stable structures includes study of the role of site-bound metal ions. Because the alkaline earth metal ions typically associated with RNA structure [most often Mg(II)] do not provide convenient spectroscopic signals, replacement with metal ions having spectroscopically useful properties has been a valuable approach. The luminescence properties of the lanthanide(III) series, in particular europium(III), have made them useful in the study of complexation with biomolecules. We review the physical, chemical, and spectroscopic characteristics of Eu(III) that contribute to its value as a probe of RNA-metal ion interactions, and examples of information obtained from studies of Eu(III) bound to small RNA stem loops. Although Eu(III) has similar site preference to Mg(II), luminescence and isothermal titration calorimetry measurements indicate that Ln(III) loses water molecules from the inner hydration sphere more readily than does Mg(II), resulting in more direct coordination between RNA and the metal ion and very different energetics of binding. In some cases, e.g., a GAAA tetraloop, binding appears to occur by a lock and key process; in the same base sequence containing certain deoxynucleoside substitutions that alter loop structure, binding appears to occur by an induced fit process.

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

Sequestering of Eu(III) by a GAAA RNA tetraloop.

The site-specific binding of metal ions maintains an important role in the structure, thermal stability, and function of folded RNA structures. RNA tetraloops of the "GNRA" family (where N = any base and R = any purine), which owe their unusual stability to base stacking and an extensive hydrogen bonding network, have been observed to bind metal ions having different chemical and geometric properties. We have used laser-induced lanthanide luminescence and isothermal titration calorimetry (ITC) to examine the metal-binding properties of an RNA stem loop of the GNRA family. Previous research has shown that a single Eu(III) ion binds the stem loop fragment in a highly dehydrated site with a K(d) of approximately 12 microM. Curve-fitting analysis of the broad luminescence excitation spectrum of Eu(III) upon complexation with the tetraloop fragment indicates the possibility of two microenvironments that do not differ in hydration number. Binding of Eu(III) to the loop was accompanied by positive enthalpic changes, consistent with energetic cost of removal of water molecules and suggesting that the binding is entropically driven. By comparison, binding of Mg(II) or Mn(II) to the RNA loop, or Eu(III) to the DNA analogue of the loop, was associated with exothermic changes, consistent with predominantly outer-sphere coordination. These results suggest specific binding, most probably involving ligands on the 5' side of the loop.