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Continued spread of chronic wasting disease (CWD) through wild cervid herds negatively impacts populations, erodes wildlife conservation, drains resource dollars, and challenges wildlife management agencies. Risk factors for CWD have been investigated at state scales, but a regional model to predict locations of new infections can guide increasingly efficient surveillance efforts. We predicted CWD incidence by county using CWD surveillance data depicting white-tailed deer (Odocoileus virginianus) in 16 eastern and midwestern US states. We predicted the binary outcome of CWD-status using four machine learning models, utilized five-fold cross-validation and grid search to pinpoint the best model, then compared model predictions against the subsequent year of surveillance data. Cross validation revealed that the Light Boosting Gradient model was the most reliable predictor given the regional data. The predictive model could be helpful for surveillance planning. Predictions of false positives emphasize areas that warrant targeted CWD surveillance because of similar conditions with counties known to harbor CWD. However, disagreements in positives and negatives between the CWD Prediction Web App predictions and the on-the-ground surveillance data one year later underscore the need for state wildlife agency professionals to use a layered modeling approach to ensure robust surveillance planning. The CWD Prediction Web App is at https://cwd-predict.streamlit.app/ ., (© 2024. The Author(s).)

While there is increasing recognition that social processes in cities like gentrification have ecological consequences, we lack nuanced understanding of the ways gentrification affects urban biodiversity. We analyzed a large camera trap dataset of mammals (>500 g) to evaluate how gentrification impacts species richness and community composition across 23 US cities. After controlling for the negative effect of impervious cover, gentrified parts of cities had the highest mammal species richness. Change in community composition was associated with gentrification in a few cities, which were mostly located along the West Coast. At the species level, roughly half (11 of 21 mammals) had higher occupancy in gentrified parts of a city, especially when impervious cover was low. Our results indicate that the impacts of gentrification extend to nonhuman animals, which provides further evidence that some aspects of nature in cities, such as wildlife, are chronically inaccessible to marginalized human populations., Competing Interests: Competing interests statement:The authors declare no competing interest.

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4' phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules' biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (© 2023 The Authors.)

Liquid-liquid phase separation is a phenomenon within biology whereby proteins can separate into dense and more dilute phases with distinct properties. Three antibodies that undergo liquid-liquid phase separation were characterized in the protein-rich and protein-poor phases. In comparison to the protein-poor phase, the protein-rich phase demonstrates more blue-shift tryptophan emissions and red-shifted amide I absorbances. Large changes involving conformational isomerization around disulfide bonds were observed using Raman spectroscopy. Amide I and protein fluorescence differences between the phases persisted to temperatures above the critical temperature but ceased at the temperature at which aggregation occurred. In addition, large changes occurred in the structural organization of water molecules within the protein-rich phase for all three antibodies. It is hypothesized that as the proteins have the same chemical potential in both phases, the protein viscosity is higher in the protein-rich phase resulting in slowed diffusion dependent protein aggregation in this phase. For all three antibodies we performed accelerated stability studies and found that the protein-rich phase aggregated at the same rate or slower than the protein-poor phase., Competing Interests: Declaration of Interests RE and CKK are employees of AstraZeneca and may own stock or stock options. The remaining authors have no interests to disclose related to this work., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Polysorbate is a key excipient included in formulations of therapeutic proteins to help prevent aggregation and surface adsorption. The stability of both polysorbate and therapeutic proteins can be compromised by oxidative degradation. In general, polysorbate is added to formulations at concentrations above the critical micelle concentration (cmc). To date, however, few experiments have quantitatively addressed the extent of extra- and intra-micellar oxidation of polysorbate in pharmaceutically relevant buffers. This study utilizes 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AAPH), a peroxyl radical-generating initiator, C11-BODIPY(581/591), a lipid peroxidation probe, and fluorescence spectroscopy to reveal that both intra- and extra-micellar oxidation proceed in pharmaceutically relevant phosphate and histidine buffers. It is further demonstrated that the relative extent of oxidation observed in the intra- and extra-micellar compartments is similar irrespective of the buffer system., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA-LNPs transition from an inverse hexagonal phase at pH values below the pK a of the cationic lipid to a lamellar phase above the pK a . At higher temperatures the mRNA-LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pK a than above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA-LNPs. The particles were larger and more prone to aggregation below the pK a . A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA-LNPs had the highest transfection efficiency and stability at pH values below the pK a . However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA-LNPs and will aid the development of related formulations., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Histone modifying enzymes regulate chromatin architecture through covalent modifications and ultimately control multiple aspects of cellular function. Disruption of histone modification leads to changes in gene expression profiles and may lead to disease. Both small molecule inhibitors and intermediary metabolites have been shown to modulate histone modifying enzyme activity although our ability to identify successful drug candidates or novel metabolic regulators of these enzymes has been limited. Using a combination of large scale in silico screens and in vivo phenotypic analysis, we identified several small molecules and intermediary metabolites with distinctive HME activity. Our approach using unsupervised learning identifies the chemical fingerprints of both small molecules and metabolites that facilitate recognition by the enzymes active sites which can be used as a blueprint to design novel inhibitors. Furthermore, this work supports the idea that histone modifying enzymes sense intermediary metabolites integrating genes, environment and cellular physiology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This work presents a combined study of time-resolved fluorescence spectroscopy and all-atom molecular dynamics simulation to investigate periodic boundary conditions' and water models' influence on the orientation dynamics and translational and rotational diffusion of peptides in solution. We have characterized the effects of solvent box size and water model choice on the dynamics of two peptide systems, NATA and WK5. Computationally, translational, and rotational diffusion and internal fluctuations are investigated through all-atom molecular dynamics simulation with two water models and different box sizes. These results are compared with time-resolved fluorescence anisotropy decay (FAD) measurements. The associated time constant and orientation dynamics from FAD measurement along the 1 L b axis provided baseline data to validate molecular dynamics simulation. The modeling results show that diffusion rates vary roughly in inverse proportion to water model viscosity, as one would expect. Corrections for finite box size are significant for translational diffusion and insignificant for rotational diffusion. This study also finds that internal dynamics described by autocorrelation functions and kinetic network models are relatively insensitive to both box size and water model properties. Our observation suggests that different peptide properties respond differently to a change in simulation conditions.Communicated by Ramaswamy H. Sarma.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius ( R h ) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its R h is in good agreement with the predicted value. The R h values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in R h between fl- and Δ1-2SRCR-LOXL2s in solution.

Picosecond time-resolved fluorescence anisotropy was used to measure the effect of denaturants and osmolytes on the reorientation dynamics of the simplest dipeptide. The solvent denaturants guanidinium hydrochloride (gdm), urea, and the osmolyte proline were used at several concentrations. Analysis of the concentration dependence of denaturants at a fixed temperature showed faster and slower reorientation time in two different denaturants at a nearly identical solvent viscosity (η). The reorientation time τ significantly deviates from Kramers' theory (τ ∝ η 1 ) in the high friction limit for guanidinium and urea with r ≈ 0.4 and r ≈ 0.6 at pH 7.2, respectively. In proline, τ is nearly proportional to η. Atomistic molecular dynamics simulations of the dipeptide in identical cosolvents showed excellent agreement with the measured rotational orientation time. The dipeptide dihedral (ϕ, ψ) isomerization times in water and 6 M urea are almost identical and significantly slower in guanidinium. If a faster and slower reorientation time can be associated with the compact and expanded shapes, the fractional viscosity dependence for guanidinium and urea may result from the fact that internal dynamics of peptides in these cosolvents involve higher and lower internal friction within the dynamic elements.

Fast kinetic experiments with dramatically improved time resolution have contributed significantly to understanding the fundamental processes in protein folding pathways involving the formation of a-helices and b-hairpin, contact formation, and overall collapse of the peptide chain. Interpretation of experimental results through application of a simple statistical mechanical model was key to this understanding. Atomistic description of all events observed in the experimental findings was challenging. Recent advancements in theory, more sophisticated algorithms, and a true long-term trajectory made way for an atomically detailed description of kinetics, examining folding pathways, validating experimental results, and reporting new findings for a wide range of molecular processes in biophysical chemistry. This review describes how optimum dimensionality reduction theory can construct a simplified coarse-grained model with low dimensionality involving a kinetic matrix that captures novel insights into folding pathways. A set of metastable states derived from molecular dynamics analysis generate an optimally reduced dimensionality rate matrix following transition pathway analysis. Analysis of the actual long-term simulation trajectory extracts a relaxation time directly comparable to the experimental results and confirms the validity of the combined approach. The application of the theory is discussed and illustrated using several examples of helix <==> coil transition pathways. This paper focuses primarily on a combined approach of time-resolved experiments and long-term molecular dynamics simulation from our ongoing work.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λ max of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N -acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λ max of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1 H, 13 C, and 1 H- 13 C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N -(2,4-dihydrocyclopenta[ b ]indol-2-yl)acetamide (structure P3a ) or a tautomer ( P3b - d ).

Approximately 100 years ago, unregulated harvest nearly eliminated white-tailed deer ( Odocoileus virginianus ) from eastern North America, which subsequently served to catalyze wildlife management as a national priority. An extensive stock-replenishment effort soon followed, with deer broadly translocated among states as a means of re-establishment. However, an unintended consequence was that natural patterns of gene flow became obscured and pretranslocation signatures of population structure were replaced. We applied cutting-edge molecular and biogeographic tools to disentangle genetic signatures of historical management from those reflecting spatially heterogeneous dispersal by evaluating 35,099 single nucleotide polymorphisms (SNPs) derived via reduced-representation genomic sequencing from 1143 deer sampled statewide in Arkansas. We then employed Simpson's diversity index to summarize ancestry assignments and visualize spatial genetic transitions. Using sub-sampled transects across these transitions, we tested clinal patterns across loci against theoretical expectations of their response under scenarios of re-colonization and restricted dispersal. Two salient results emerged: (A) Genetic signatures from historic translocations are demonstrably apparent; and (B) Geographic filters (major rivers; urban centers; highways) now act as inflection points for the distribution of this contemporary ancestry. These results yielded a statewide assessment of contemporary population structure in deer as driven by historic translocations as well as ongoing processes. In addition, the analytical framework employed herein to effectively decipher extant/historic drivers of deer distribution in Arkansas is also applicable for other biodiversity elements with similarly complex demographic histories., Competing Interests: None declared., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

The rapid growth in technological advances and quantity of scientific data over the past decade has led to several challenges including data storage and analysis. Accurate models of complex datasets were previously difficult to develop and interpret. However, improvements in machine learning algorithms have since enabled unparalleled classification and prediction capabilities. The application of machine learning can be seen throughout diverse industries due to their ease of use and interpretability. In this review, we describe popular machine learning algorithms and highlight their application in pharmaceutical protein development. Machine learning models have now been applied to better understand the nonlinear concentration dependent viscosity of protein solutions, predict protein oxidation and deamidation rates, classify sub-visible particles and compare the physical stability of proteins. We also applied several machine learning algorithms using previously published data and describe models with improved predictions and classification. The authors hope that this review can be used as a resource to others and encourage continued application of machine learning algorithms to problems in pharmaceutical protein development., (Published by Elsevier Inc.)

A synthetic peptide, K-PLP, consisting of 11-unit poly-lysine (K11) linked via polyethylene glycol (PEG) to proteolipid protein epitope (PLP) was synthesized, characterized, and evaluated for efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE) induced by PLP. K-PLP was designed to mimic the cationic nature of the relapsing-remitting multiple sclerosis treatment, glatiramer acetate (GA). With a pI of ~10, GA is able to form visible aggregates at the site of injection via electrostatic interactions with the anionic extracellular matrix. Aggregation further facilitates the retention of GA at the site of injection and draining lymph nodes, which may contribute to its mechanism of action. K-PLP with a pI of ~11, was found to form visible aggregates in the presence of glycosaminoglycans and persist at the injection site and draining lymph nodes in vivo , similar to GA. Additionally, EAE mice treated with K-PLP showed significant inhibition of clinical symptoms compared to free poly-lysine and to PLP, which are the components of K-PLP. The ability of the poly-lysine motif to retain PLP at the injection site, which increased the local exposure of PLP to immune cells may be an important factor affecting drug efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Song, Griffin, Larson, Christopher, Middaugh and Berkland.)

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17-36 a.a. "RG1" epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic-wasting disease (CWD) is a prion-derived fatal neurodegenerative disease that has affected wild cervid populations on a global scale. Susceptibility has been linked unambiguously to several amino acid variants within the prion protein gene ( PRNP ). Quantifying their distribution across landscapes can provide critical information for agencies attempting to adaptively manage CWD. Here we attempt to further define management implications of PRNP polymorphism by quantifying the contemporary geographic distribution (i.e., phylogeography) of PRNP variants in hunter-harvested white-tailed deer (WTD; Odocoileus virginianus , N = 1433) distributed across Arkansas (USA), including a focal spot for CWD since detection of the disease in February 2016. Of these, PRNP variants associated with the well-characterized 96S non-synonymous substitution showed a significant increase in relative frequency among older CWD-positive cohorts. We interpreted this pattern as reflective of a longer life expectancy for 96S genotypes in a CWD-endemic region, suggesting either decreased probabilities of infection or reduced disease progression. Other variants showing statistical signatures of potential increased susceptibility, however, seemingly reflect an artefact of population structure. We also showed marked heterogeneity across the landscape in the prevalence of 'reduced susceptibility' genotypes. This may indicate, in turn, that differences in disease susceptibility among WTD in Arkansas are an innate, population-level characteristic that is detectable through phylogeographic analysis.

Smallpox, a contagious and deadly disease caused by variola virus, was eradicated by a strategy that included vaccination with vaccinia virus, a live-virus vaccine. Because the threat of bioterrorism with smallpox persists and infections with zoonotic poxvirus infections like monkeypox continue, and there may be a time when an alternative vaccine platform is needed, recombinant-subunit vaccine strategies for poxviruses have been pursued. Our prior work focused on understanding the immune responses generated to vaccine-formulations containing the virus protein L1. In this work, we examine vaccine-formulations with additional key protein targets: A33 and B5 (components of the extracellular virus) and another protein on the mature virus (A27) adjuvanted with aluminum hydroxide (AH) with and without CpG- oligonucleotide. Each vaccine was formulated to allow either adsorption or non-adsorption of the protein (and CpG) to AH. Mice given a prime and single boost produced long-lasting antibody responses. A second boost (given ~5-months after the first) further increased antibody titers. Similar to our prior findings with L1 vaccine-formulations, the most protective A33 vaccine-formulations included CpG, resulted in the generation of IgG2a-antibody responses. Unlike the prior findings with L1 (where formulations that adsorbed both the protein and the CpG to AH resulted in 100% survival after challenge and minimal weight loss), the AH-adsorption status of A33 and CpG did not play as important a role, since both AH-adsorbed and non-adsorbed groups lost weight after challenge and had similar survival. Vaccination with B5-formulations gave different results. While CpG-containing formulations were the only ones that generated IgG2a-antibody responses, the vaccine-formulation that adsorbed B5 to AH (without CpG) was as equally effective in protecting mice after challenge. These results indicate that the mechanism of how antibodies against A33 and B5 protect differ. The data also show the complexity of designing optimized vaccine-formulations containing multiple adjuvants and recombinant protein-based antigens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aberrant splicing and protein interaction of Ras binding domain (RBD) are associated with melanoma drug resistance. Here, cobalt or nickel doped zinc oxide (ZnO) physiometacomposite (PMC) materials bind to RNA and peptide shown by Ninhydrin staining, UV-vis, Fourier transform infrared, and circular dichroism spectroscopy. PMCs deliver splice switching oligomer (SSO) into melanoma cells or 3-D tumor spheroids shown by flow cytometry, fluorescence microscopy, and bioluminescence. Stability in serum, liver, or tumor homogenate up to 48 h and B16F10 melanoma inhibition ≥98-99% is shown. These data suggest preclinical potential of PMC for delivery of SSO, RBD, or other nucleic acid therapeutic and anticancer peptides.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Polysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. We studied the 2 main pathways of polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis, and oxidation. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, whereas the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of nondegraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc 2 Man (8+n) [n = 0-4]), Man 5 (GlcNAc 2 Man 5 ), GlcNAc 1 , and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max  = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn 297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc 1 > Man 5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man 5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc 1 IgG4-Fc. The affinities measured for the HM and Man 5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na 2 SO 4 ), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride > trimethylphenylammonium iodide > tryptophan amide hydrochloride > ethanol as measured by various biophysical techniques. Conversely, addition of Na 2 SO 4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Oxidation of therapeutic proteins (TPs) can lead to changes in their pharmacokinetics, biological activity and immunogenicity. Metal impurities such as iron are known to increase oxidation of TPs, but nanoparticulate metals have unique physical and chemical properties compared to the bulk material or free metal ions. Iron oxide nanoparticles (IONPs) may originate from equipment used in the manufacturing of TPs or from needles during injection. In this study, the impact of IONPs on oxidation of a model protein, rat growth hormone (rGH), was investigated under chemical stress. Hydrogen peroxide (H 2 O 2 )- and 2,2'-azobis (2-methylpropionamidine) dihydrochloride oxidized methionine residues of rGH, but unexpectedly, oxidation was suppressed in the presence of IONPs compared to a phosphate buffer control. Fourier transform infrared spectroscopy indicated splitting of the α-helical absorbance band in the presence of IONPs, whereas circular dichroism spectra showed a reduced α-helical contribution with increasing temperature for both rGH and rGH-IONP mixtures. The results collectively indicate that IONPs can increase the chemical stability of rGH by altering the kinetics and preference of amino acid residues that are oxidized, although the changes in protein secondary structure by IONPs may lead to alterations of physical stability., (Copyright © 2019. Published by Elsevier Inc.)

Type III secretion systems are used by many Gram-negative bacteria to inject effector proteins into eukaryotic cells to subvert their normal activities. Structurally conserved portions of the type III secretion apparatus include a: basal body located within the bacterial envelope; an exposed needle with tip complex that delivers effectors across the target cell membrane; and cytoplasmic sorting platform that selects cargo and powers secretion. While structurally conserved, the individual proteins that make up this nanomachine are typically not interchangeable though they do tend to fall into families. Here we selected a single domain from the inner membrane ring of the basal body from six different type III secretion systems (called SctD using a proposed unifying nomenclature). The selected domain creates an integral interface between the basal body and the sorting platform. Therefore, it represents a pivotal point between two distinct assemblies. All six protein domains possess a structural motif called a forkhead-associated-like (FHA-like) domain but differ greatly in their sequences and solution behaviors. These differences are used here to define family boundaries for these FHA-like domains. The data parallel, though not precisely, family boundaries defined by other proteins within the apparatus and by phylogenetic analysis. Ultimately, differences in the families are likely to reflect differences in the activities of these type III secretion systems or the host niches in which these pathogens are found., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Autoimmune diseases are believed to be highly dependent on loss of immune tolerance to self-antigens. Currently, no treatments have been successful clinically in inducing autoantigen-specific tolerance, including efforts to utilize antigen-specific immunotherapy (ASIT) to selectively correct the aberrant autoimmunity. Soluble antigen arrays (SAgAs) represent a novel autoantigen delivery system composed of a linear polymer, hyaluronic acid (HA), displaying multiple copies of conjugated autoantigen. We have previously reported that soluble antigen arrays displaying proteolipid peptide (SAgA PLP ) induced tolerance to this specific multiple sclerosis (MS) autoantigen. Utilizing SAgA technology, we have developed a new ASIT as a possible type 1 diabetes (T1D) therapeutic by conjugating human insulin to HA, known as soluble antigen array insulin (SAgA Ins ). Three types were synthesized, low valency lv SAgA Ins (2 insulins per HA), medium valency mv SAgA Ins (4 insulins per HA), and, high valency hv SAgA Ins (9 insulins per HA), to determine if valency differentially modulates the ex vivo activity of insulin-binding B cells (IBCs). Extensive biophysical characterization was performed for the SAgA molecules. SAgA Ins molecules were successfully used to affect the biologic activity of IBCs by inducing desensitization of the B cell antigen receptors (BCR). SAgA Ins bound specifically to insulin-reactive B cells without blocking epitopes recognized by antibodies against the Fc regions of membrane immunoglobulin or CD79 transducer components of the BCR. Preincubation of IBCs (125Tg) with SAgA Ins , but not HA alone, rendered the IBCs refractory to restimulation. SAgA Ins induced a decrease in BCR expression and IP3R-mediated intracellular calcium release. Surprisingly, SAgA Ins binding to BCR on the surface of IBCs induced the observed effects at both high and low SAgA Ins valency. Future studies aim to test the effects of SAgA Ins on disease progression in the VH125.NOD mouse model of T1D.

Oral administration is advantageous compared to the commonly used parenteral administration for local therapeutic uses of biologics or mucosal vaccines, since it can specifically target the gastrointestinal (GI) tract. It offers better patient compliance, even though the general use of such a delivery route is often limited by potential drug degradation in the GI tract and poor absorption. Using bovine serum albumin (BSA) and lysozyme as two model proteins, we studied their solid-state properties, mechanical properties, and tabletability as well as effects of compaction pressure, particle size, and humidity on protein degradation. It was found that BSA and lysozyme are highly hygroscopic, and their tablet manufacturability (powder caking, punch sticking, and tablet lamination) is sensitive to the humidity. BSA and lysozyme exhibited high plasticity and excellent tabletability and remained amorphous at high pressure and humidity. As for protein stability, lysozyme was resistant to high pressure (up to 300 MPa) and high humidity (up to 93%). In contrast, BSA underwent aggregation upon compression, an effect that was more pronounced for smaller BSA particles. High humidity accelerated the aggregation of BSA during incubation, but it did not further synergize with mechanical stress to induce protein degradation. Thus, compression can potentially induce protein aggregation, but this effect is protein-dependent. Therefore, strategies (e.g., the use of excipients, optimized manufacturing processes) to inhibit protein degradation should be explored before their tablet dosage form development.

Glatiramer acetate (GA) is widely prescribed for the treatment of relapsing-remitting multiple sclerosis, however, the mechanism of action is still not fully understood. We investigated the structural properties of GA and examined alterations to the drug upon injection into the subcutaneous space. First, a variety of biophysical characterization techniques were employed to characterize GA in solution. GA was found to exist as alpha helices in solution with a hydrodynamic radius of ~3 nm in size. To simulate GA behavior at the site of injection, GA was injected into a solution of 1.5 MDa hyaluronic acid (HA). Visible aggregates were observed immediately upon injection and subsequent testing indicated aggregation was driven by electrostatic interactions between the positively-charged GA and negatively-charged HA. In vivo testing confirmed GA formed spherical particles in the nano- to micrometer size range, suggesting this mechanism contributes to persistence at the injection site and in draining lymph nodes. The aggregates were found to associate with glycosaminoglycans, suggesting an electrostatic mechanism of induced aggregation like the simulated injection. These novel observations may help explain the complex immunomodulatory mechanisms of GA and adverse injection site reactions seen in patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (V H Hs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The V H Hs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot ("cluster 3") located in close proximity to RTA's active site. HX-MS analysis revealed that the 21 V H Hs recognized four distinct epitope subclusters (3.1-3.4). Sixteen of the 21 V H Hs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three V H Hs grouped within subcluster 3.2, encompassing a-helices C and G, plus α-helix B. The single V H H in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole V H H defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 V H Hs within subclusters 3.1-3.3 physically occlude RTA's active site cleft, while the single antibody in subcluster 3.4 associates on the active site's upper rim., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from -80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at -20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed., (Copyright © 2018 American Pharmacists Association®. All rights reserved.)

We have used hydrogen exchange-mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Ricin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report, we describe five new mouse mAbs directed against an immunodominant region, so-called epitope cluster II, on the surface of ricin's ribosome-inactivating enzymatic subunit A (RTA). The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10× LD 50 ricin challenge by injection. Only three of the mAbs (LE4, PH12, and TB12) afforded protection over the 7-d study period. Neither binding affinity nor in vitro toxin-neutralizing activity could fully account for LE4, PH12, and TB12's potent in vivo activity relative to the other six mAbs. However, epitope mapping studies by hydrogen exchange-mass spectrometry revealed that LE4, PH12, and TB12 shared common contact points on RTA corresponding to RTA α-helices D and E and β-strands d and e located on the back side of RTA relative to the active site. The other six mAbs recognized overlapping epitopes on RTA, but none shared the same hydrogen exchange-mass spectrometry profile as LE4, PH12, and TB12. A high-density competition ELISA with a panel of ricin-specific, single-domain camelid Abs indicated that even though LE4, PH12, and TB12 make contact with similar secondary motifs, they likely approach RTA from different angles. These results underscore how subtle differences in epitope specificity can significantly impact Ab functionality in vivo. ImmunoHorizons , 2018, 2: 262-273.

Lumazine synthase (LS) is a family of enzyme involved in the penultimate step in the biosynthesis of riboflavin. Its enzymatic mechanism has been well defined, and many LS structures have been solved using X-ray crystallography or cryoelectron microscopy. LS is composed of homooligomers, which vary in size and subunit number, including pentamers, decamers, and icosahedral sixty-mers, depending on its species of origin. Research on LS has expanded beyond the initial focus on its enzymatic function to properties related to its oligomeric structure and exceptional conformational stability. These attributes of LS systems have now been repurposed for use in various biomedical fields. This review primarily focuses on the applications of LS as a flexible vaccine presentation system. Presentation of antigens on the surface of LS results in a high local concentration of antigens displayed in an ordered array. Such repetitive structures enable the cross-linking of B-cell receptors and result in strong immune responses through an avidity effect. Potential issues with the use of this system and corresponding solutions are also discussed with the objective of improved utilization of the LS system in vaccine development., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The effects of 2 squalene-based emulsion adjuvant systems (MedImmune emulsion 0 [ME.0] and Stable Emulsion [SE]) on the structure and stability of the recombinant protein antigen alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection, were investigated using Fourier-transform infrared spectroscopy and both steady-state and time-resolved intrinsic fluorescence spectroscopy as well as differential scanning calorimetry (DSC). A component study, performed to identify the effects of the individual emulsion's components, showed negligible interactions between AT and ME.0. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a significant blue shift in the fluorescence spectra relative to that of AT in solution. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. This work successfully characterized the biophysical properties of AT in the presence of 2 emulsion adjuvants including a component study to rationalize how emulsion components affect protein antigen stability., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Climate change will affect stream systems in numerous ways over the coming century. Globally, streams are expected to experience changes in temperature and flow regime. Previous work has indicated that these changes will likely affect fish distributions, but little work has been conducted examining population level effects of climate change on warmwater fish at the warmest portion of their range. We model several potential climate change-related stressors and the resulting effects on smallmouth bass Micropterus dolomieu populations in the Buffalo National River, Arkansas, USA, located near the southern extent of smallmouth bass range. Smallmouth bass are a popular recreational fish in the region and angler harvest likely contributes substantially to annual mortality. We created a simulation model parameterized with data collected from the Buffalo National River to evaluate the relative importance of climate stressors and angler harvest on smallmouth bass populations. Our simulations suggest that increases in springtime temperature and reductions in river discharge during the spawning period could increase recruitment, resulting in increases in adult abundance (8% higher). However, when increased flooding and drought probabilities are considered, our model indicates the Buffalo National River could experience large reductions in adult smallmouth bass abundance (≥50% decline) and increased probability of extinction compared to present levels. Simulations showed that harvest reduction could be a viable strategy to reduce the negative effects of climate change, but that even with complete closure of harvest, smallmouth bass population levels would still be well below present abundance (46% lower than present). Efforts to reduce flooding and drought effects related to climate change in the Buffalo National River could help offset the predicted reduction in the smallmouth bass population., Competing Interests: The authors have declared that no competing interests exist.

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's β-strand h (732 Å2 buried surface area; BSA), along with limited engagement with α-helix D (90 Å2) and α-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with α-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics., (© 2018 The Protein Society.)