Creating an Integrated Coaching Culture in Higher Education.

In higher education, it is a common ask to do more with less while delivering high-quality, holistic service to students. Coaching has been shown to produce significant gains in strengthening self-efficacy, improving GPA, and increasing retention through graduation (Alzen et al., 2021; Capstick et al., 2019; Catchings, 2014; Grover & Furnham, 2016; Losch et al., 2016), therefore making it a logical program to target for growth. To expand the impact of the University of Kentucky's academic coaching program, in 2020, leadership modified the Appreciative Academic Coaching framework (Bradley & Reynolds, 2021) into Integrated Success Coaching with the intent to build a coaching culture across campus. This modification created a two-pronged approach to serving students, faculty, and staff across our campus: (a) training for professionally certified International Coaching Federation (ICF) coaches across six domains, including academic life, career, finances, wellness, leadership, and identity (e.g., First Gen) to directly serve students, and (b) training in foundational coaching skills for faculty, staff, and student leaders to incorporate into their daily practice. The evolution of this coaching model has allowed for holistic support of students and immersive coaching values and practices for faculty, staff, and student leaders that have led to improved retention and better GPA outcomes for students on probation and a culture of coaching care among faculty, staff, and students.

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers.

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4'-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

STEM Through Authentic Research and Training Program (START) for Underrepresented Communities: Adapting to the COVID-19 Pandemic.

The STEM Through Authentic Research and Training (START) Program is a new program integrating academic, social, and professional experiences, in the theme of exomedicine, to build a pipeline into college for first generation and traditionally underrepresented students by providing year-round authentic opportunities and professional development for high school students and teachers. In response to the COVID-19 pandemic, the START Program has worked with the local Fayette County public school and community partners to provide content to over 300 students through: virtual laboratory tours with community partner Space Tango, "meet a scientist" discussions, and online near-peer student demonstrations aimed at making the practice of STEM disciplines approachable. Furthermore, the START Program has partnered with Higher Orbits to provide at-home, space-themed learning kits for students to develop teamwork, communication, and STEM principles while engaging in online content with teachers, professionals, and astronauts. Finally, the START Program has moved its training platforms online, including receiving College Reading and Learning Association (CRLA) Peer Educator accreditation for our near-peer mentoring and coaching training. As a result, the START Program is better positioned to address this critical need in STEM education, while reaching more students in the community than possible with face-to-face interactions alone.

GDNF revisited: A novel mammalian cell-derived variant form of GDNF increases dopamine turnover and improves brain biodistribution.

Parkinson's disease (PD) is a disorder affecting dopamine neurons for which there is no cure. Glial cell line-derived neurotrophic factor (GDNF) and the closely related protein neurturin are two trophic factors with demonstrated neuroprotective and neurorestorative properties on dopamine neurons in multiple animal species. However, GDNF and neurturin Phase-2 clinical trials have failed to demonstrate a significant level of improvement over placebo controls. Insufficient drug distribution in the brain parenchyma has been proposed as a major contributing factor for the lack of clinical efficacy in the Phase-2 trial patients. To address this issue, a novel mammalian cell-derived variant form of GDNF (GDNFv) was designed to promote better tissue distribution by reducing its heparin binding to the extracellular matrix and key amino acids were substituted to enhance its chemical stability. Administration of this fully glycosylated GDNFv in the normal rat striatum increased dopamine turnover and produced significantly greater brain distribution than E. coli-produced wildtype GDNF (GDNFwt). Intrastriatal GDNFv also protected midbrain dopamine neuron function in 6-hydroxydopamine-lesioned rats. Studies conducted in normal adult rhesus macaques support that GDNFv was well tolerated in all animals and demonstrated a greater volume of distribution than GDNFwt in the brain following intrastriatal infusion. Importantly, favorable physiological activity of potential therapeutic value was maintained in this variant trophic factor with significant target activation in GDNFv recipients as indicated by dopamine turnover modulation. These data suggest that GDNFv may be a promising drug candidate for the treatment of PD. Additional studies are needed in non-human primates with dopamine depletion. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease.

BACKGROUND: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. NEW METHOD: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. RESULTS: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300µg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrease in DA turnover in the lesioned striatum; and an increased sparing of tyrosine hydroxylase (TH) positive (+) neurons in a specific sub-region of the lesioned substantia nigra pars compacta (SNpc). Finally, tracer studies showed (125)I-DNSP-11 distributed diffusely throughout the brain, including the striatum and SN, as quickly as 30min after a single intranasal dose. COMPARISON WITH EXISTING METHODS: The results of bilateral intranasal administration of DNSP-11 are compared to our unilateral single infusion studies to the brain in rats. CONCLUSIONS: These studies support that DNSP-11 can be delivered intranasally and maintain its neuroactive properties in both normal rats and in a unilateral 6-OHDA rat model of PD.

High-quality combinatorial protein libraries using the binary patterning approach.

Protein combinatorial libraries have become a platform technology for exploring protein sequence space for novel molecules for use in research, synthetic biology, biotechnology, and medicine. To expedite the isolation of proteins with novel/desired functions using screens and selections, high-quality approaches that generate protein libraries rich in folded and soluble structures are desirable for this goal. The binary patterning approach is a protein library design method that incorporates elements of both rational design and combinatorial diversity to specify the arrangement of polar and nonpolar amino acid residues in the context of a desired, folded tertiary structure template. An overview of the considerations necessary to design and construct binary patterned libraries of de novo and natural proteins is presented.

Dynamic changes in dopamine neuron function after DNSP-11 treatment: effects in vivo and increased ERK 1/2 phosphorylation in vitro.

Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and D-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.

Convection enhanced delivery of different molecular weight tracers of gadolinium-tagged polylysine.

Convection enhanced delivery (CED) is a powerful method of circumventing the blood-brain barrier (BBB) to deliver therapeutic compounds directly to the CNS. While inferring the CED distribution of a therapeutic compound by imaging a magnetic resonance (MR)-sensitive tracer has many advantages, however how the compound distribution is affected by the features of the delivery system, its target tissue, and its molecular properties, such as its binding characteristics, charge, and molecular weight (MW) are not fully understood. We used MR imaging of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)-tagged polylysine compounds of various MW, in vitro and in vivo, to measure the dependence of compounds MW on CED distribution. For the in vitro studies, the correlation between volume of distribution (Vd) as a function of MW was determined by measuring the T1 of the infused tracers, into 0.6% agarose gels through a multiport catheter. The compounds distributed in the gels inversely proportional to their MW, consistent with convection and unobstructed diffusion through a porous media. For the in vivo studies, Gd-DTPA tagged compounds were infused into the non-human primate putamen, via an implanted multiport catheter connected to a MedStream™ pump, programmed to deliver a predetermined volume with alternating on-off periods to take advantage of the convective and diffusive contributions to Vd. Unlike the gel studies, the higher MW polylysine-tracer infusions did not freely distribute from the multiport catheter in the putamen, suggesting that distribution was impeded by other properties that should also be considered in future tracer design and CED infusion protocols.

Utilization of a calmodulin lysine methyltransferase co-expression system for the generation of a combinatorial library of post-translationally modified proteins.

By successfully incorporating sequence diversity into proteins, combinatorial libraries have been a staple technology used in protein engineering, directed evolution, and synthetic biology for generating proteins with novel specificities and activities. However, these approaches mostly overlook the incorporations of post-translational modifications, which nature extensively uses for modulating protein activities in vivo. As an initial step of incorporating post-translational modifications into combinatorial libraries, we present a bacterial co-expression system, utilizing a recently characterized calmodulin methyltransferase (CaM KMT), to trimethylate a combinatorial library of the calmodulin central linker region. We show that this system is robust, with the successful over-expression and post-translational modification performed in Escherichia coli. Furthermore we show that trimethylation differentially affected the conformational dynamics of the protein upon the binding of calcium, and the thermal stability of the apoprotein. Collectively, these data support that when applied to an appropriately designed protein library scaffold, CaM KMT is able to produce a post-translationally modified library of protein sequences, thus providing a powerful tool for future protein library designs and constructions.

Domain-swapped dimeric structure of a stable and functional de novo four-helix bundle protein, WA20.

To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

Active site mutations change the cleavage specificity of neprilysin.

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563) and Ser(546). Among the mutants studied in detail we observed changes in their activity towards leucine(5)-enkephalin, insulin B chain, and amyloid ß(1-40). For example, NEP(F563I) displayed an increase in preference towards cleaving leucine(5)-enkephalin relative to insulin B chain, while mutant NEP(S546E) was less discriminating than neprilysin. Mutants NEP(F563L) and NEP(S546E) exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40) as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence.

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.

De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth.

A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6) de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.

Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker.

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: all library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

Dopamine neuron stimulating actions of a GDNF propeptide.

BACKGROUND: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. METHODS AND FINDINGS: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. CONCLUSIONS: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor.

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins.

To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4-helix bundle proteins. This collection of novel proteins can be considered an "artificial superfamily" of helical bundles. The superfamily of 102-residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library were screened for a range of biological functions including heme binding and peroxidase, esterase, and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins-which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)-can provide rudimentary activities and serve as a "feedstock" for evolution.

Protein design by binary patterning of polar and nonpolar amino acids.

The design of large libraries of well-folded de novo proteins is a powerful approach toward the ultimate goal of producing proteins with novel structures and functions for use in industry or medicine. A method for library design that incorporates both rational design and combinatorial diversity relies on the "binary patterning" of polar and nonpolar amino acids. Binary patterning is based on the premise that the appropriate arrangement of polar and nonpolar residues can direct a polypeptide chain to fold into amphipathic elements of secondary structure that anneal together to form a desired tertiary structure. A designed binary pattern exploits the periodicities inherent in protein secondary structure, and allows the identity of the side chain at each polar and nonpolar position to be varied combinatorially. This chapter provides an overview of the considerations necessary to use binary patterning to design libraries of novel proteins.

De novo proteins from binary-patterned combinatorial libraries.

Combinatorial libraries of well-folded de novo proteins can provide a rich source of reagents for the isolation of novel molecules for biotechnology and medicine. To produce libraries containing an abundance of well-folded sequences, we have developed a method that incorporates both rational design and combinatorial diversity. Our method specifies the "binary patterning" of polar and nonpolar amino acids, but allows combinatorial diversity of amino acid side chains at each polar and nonpolar site in the sequence. Protein design by binary patterning is based on the premise that the appropriate arrangement of polar and nonpolar residues can direct a polypeptide chain to fold into amphipathic elements of secondary structures, which anneal together to form a desired tertiary structure. A designed binary pattern exploits the periodicities inherent in protein secondary structure, while allowing the identity of the side chain at each polar and non-polar position to be varied combinatorially. This chapter provides an overview of the considerations necessary to design binary patterned libraries of novel proteins.

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.