Influence of Peptoid Sequence on the Mechanisms and Kinetics of 2D Assembly.

Two-dimensional (2D) materials have attracted intense interest due to their potential for applications in fields ranging from chemical sensing to catalysis, energy storage, and biomedicine. Recently, peptoids, a class of biomimetic sequence-defined polymers, have been found to self-assemble into 2D crystalline sheets that exhibit unusual properties, such as high chemical stability and the ability to self-repair. The structure of a peptoid is close to that of a peptide except that the side chains are appended to the amide nitrogen rather than the α carbon. In this study, we investigated the effect of peptoid sequence on the mechanism and kinetics of 2D assembly on mica surfaces using in situ AFM and time-resolved X-ray scattering. We explored three distinct peptoid sequences that are amphiphilic in nature with hydrophobic and hydrophilic blocks and are known to self-assemble into 2D sheets. The results show that their assembly on mica starts with deposition of aggregates that spread to establish 2D islands, which then grow by attachment of peptoids, either monomers or unresolvable small oligomers, following well-known laws of crystal step advancement. Extraction of the solubility and kinetic coefficient from the dependence of the growth rate on peptoid concentration reveals striking differences between the sequences. The sequence with the slowest growth rate in bulk and with the highest solubility shows almost no detachment; i.e., once a growth unit attaches to the island edge, there is almost no probability of detaching. Furthermore, a peptoid sequence with a hydrophobic tail conjugated to the final carboxyl residue in the hydrophilic block has enhanced hydrophobic interactions and exhibits rapid assembly both in the bulk and on mica. These assembly outcomes suggest that, while the π-π interactions between adjacent hydrophobic blocks play a major role in peptoid assembly, sequence details, particularly the location of charged groups, as well as interaction with the underlying substrate can significantly alter the thermodynamic stability and assembly kinetics.

Development of a Systematic and Extensible Force Field for Peptoids (STEPs).

Peptoids (N-substituted glycines) are a class of biomimetic polymers that have attracted significant attention due to their accessible synthesis and enzymatic and thermal stability relative to their naturally occurring counterparts (polypeptides). While these polymers provide the promise of more robust functional materials via hierarchical approaches, they present a new challenge for computational structure prediction for material design. The reliability of calculations hinges on the accuracy of interactions represented in the force field used to model peptoids. For proteins, structure prediction based on sequence and de novo design has made dramatic progress in recent years; however, these models are not readily transferable for peptoids. Current efforts to develop and implement peptoid-specific force fields are spread out, leading to replicated efforts and a fragmented collection of parameterized sidechains. Here, we developed a peptoid-specific force field containing 70 different side chains, using GAFF2 as starting point. The new model is validated based on the generation of Ramachandran-like plots from DFT optimization compared against force field reproduced potential energy and free energy surfaces as well as the reproduction of equilibrium cis/trans values for some residues experimentally known to form helical structures. Equilibrium cis/trans distributions (Kct) are estimated for all parameterized residues to identify which residues have an intrinsic propensity for cis or trans states in the monomeric state.

A molecular dynamics investigation of N-glycosylation effects on T-cell receptor kinetics.

The binding interaction between the T-cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) is modulated by several factors (known and unknown), however, investigations into effects of glycosylation are limited. A fully glycosylated computational model of the TCR bound to the pMHC is developed to investigate the effects of glycosylation on dissociation kinetics from the pMHC. Here, we examine the effects of N-glycosylation on TCR-pMHC bond strength using steered molecular dynamic simulations. N-glycosylation is a post-translational modification that adds sugar moieties to molecules and can modulate the activity of several immune molecules. Using a TCR-pMHC pair found in melanoma as a case study, our study demonstrates that N-glycosylation of the TCR-pMHC alters the proteins' conformation; increases the bond lifetime; and increases the number of hydrogen bonds and Lennard-Jones Contacts involved in the TCR-pMHC bond. We find that weak glycan-protein or glycan-glycan interactions impact the equilibrated structure of the TCR and pMHC leading to an increase in the overall bond strength of the TCR-pMHC complex including the duration and energetic strength under constant load. These results indicate that N-glycosylation plays an important role in the TCR-pMHC bond and should be considered in future computational and experimental studies.Communicated by Ramaswamy H. Sarma.

Exogenous polyunsaturated fatty acids (PUFAs) influence permeability, antimicrobial peptide resistance, biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida.

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.

Simulated Docking Predicts Putative Channels for the Transport of Long-Chain Fatty Acids in Vibrio cholerae.

Fatty acids (FA) play an important role in biological functions, such as membrane homeostasis, metabolism, and as signaling molecules. FadL is the only known protein that uptakes long-chain fatty acids in Gram-negative bacteria, and this uptake has traditionally been thought to be limited to fatty acids up to 18 carbon atoms in length. Recently however, it was found Vibrio cholerae has the ability to uptake fatty acids greater than 18 carbon atoms and this uptake corresponds to bacterial survivability. Using E. coli's FadL as a template, V. cholerae FadL homologs vc1042, vc1043, and vca0862 have been computationally folded, simulated on an atomistic level using Molecular Dynamics, and docked in silico to analyze the FadL transport channels. For the vc1042 and vc1043 homologs, these transport channels have more structural accommodations for the many rigid unsaturated bonds of long-chain polyunsaturated fatty acids, while the vca0862 homolog was found to lack transport channels within the signature beta barrel of FadL proteins.

Transcriptional dynamics of colorectal cancer risk associated variation at 11q23.1 correlate with tuft cell abundance and marker expression in silico.

Colorectal cancer (CRC) is characterised by heritable risk that is not well understood. Heritable, genetic variation at 11q23.1 is associated with increased colorectal cancer (CRC) risk, demonstrating eQTL effects on 3 cis- and 23 trans-eQTL targets. We sought to determine the relationship between 11q23.1 cis- and trans-eQTL target expression and test for potential cell-specificity. scRNAseq from 32,361 healthy colonic epithelial cells was aggregated and subject to weighted gene co-expression network analysis (WGCNA). One module (blue) included 19 trans-eQTL targets and was correlated with POU2AF2 expression only. Following unsupervised clustering of single cells, the expression of 19 trans-eQTL targets was greatest and most variable in cluster number 11, which transcriptionally resembled tuft cells. 14 trans-eQTL targets were found to demarcate this cluster, 11 of which were corroborated in a second dataset. Intra-cluster WGCNA and module preservation analysis then identified twelve 11q23.1 trans-eQTL targets to comprise a network that was specific to cluster 11. Finally, linear modelling and differential abundance testing showed 11q23.1 trans-eQTL target expression was predictive of cluster 11 abundance. Our findings suggest 11q23.1 trans-eQTL targets comprise a POU2AF2-related network that is likely tuft cell-specific and reduced expression of these genes correlates with reduced tuft cell abundance in silico.

Production of novel SARS-CoV-2 Spike truncations in Chinese hamster ovary cells leads to high expression and binding to antibodies.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Impact of Surface Polarity on Lipid Assembly under Spatial Confinement.

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.

SARS-CoV-2 spike binding to ACE2 is stronger and longer ranged due to glycan interaction.

Highly detailed steered molecular dynamics simulations are performed on differently glycosylated receptor binding domains of the severe acute respiratory syndrome coronavirus-2 spike protein. The binding strength and the binding range increase with glycosylation. The interaction energy rises very quickly when pulling the proteins apart and only slowly drops at larger distances. We see a catch-slip-type behavior whereby interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode is hydrogen bonds, but Lennard-Jones and electrostatic interactions are relevant as well.

A survey of emergency and critical care veterinarians regarding IV fluid bolus therapy and monitoring practices in small animals.

OBJECTIVE: To determine how veterinary emergency and critical care clinicians define IV fluid bolus therapy (FBT) and what constitutes a positive response to a fluid bolus. DESIGN: Online survey of 222 emergency and critical care veterinarians between December 17, 2018, and March 1, 2019. INTERVENTIONS: An online survey was provided to diplomates of the American College of Veterinary Emergency and Critical Care (ACVECC), residents of ACVECC-approved training programs, as well as house officers and emergency clinicians of a corporate multicenter emergency and specialty care veterinary hospital. The survey investigated the administration of various crystalloid, colloid, and blood products for FBT, as well as expected physiological responses. MEASUREMENTS AND MAIN RESULTS: The majority of respondents considered balanced isotonic crystalloids appropriate for FBT (220/222 [99.1%]). Respondents showed greater variability in acceptance of 0.9% sodium chloride (105/222 [47.30%]), hypertonic (3-7%) sodium chloride (131/222 [59.01%]), and hydroxyethyl starch solutions (90/222 [40.54%]). Most respondents did not consider physiological plasma (44/222 [19.82%]) an appropriate choice. The most commonly used parameters for monitoring FBT responses were heart rate (220/222 [99.10%]), blood pressure (217/222 [97.75%]), capillary refill time (192/222 [86.49%]), lactate (181/222 [81.53%]), pulse pressure (151/222 [68.02%]), and rectal temperature (145/222 [65.32%]). The majority of respondents perceived that 0-20% (165/222 [74.32%]) of hypotensive patients are nonresponsive to FBT. CONCLUSIONS: Small animal emergency and critical care clinicians favored balanced isotonic electrolyte solutions and hypertonic sodium chloride solutions for FBT over other options. When monitoring responses to FBT, heart rate, blood pressure, capillary refill time, and plasma lactate were among the most commonly monitored parameters, and there was a lack of familiarity with others. Despite the widespread use of FBT, these findings outline the need for further prospective clinical trials regarding the ideal fluid type and rate, as well as the appropriate responses to FBT.

Perspectives on Individual Animal Identification from Biology and Computer Vision.

Identifying individual animals is crucial for many biological investigations. In response to some of the limitations of current identification methods, new automated computer vision approaches have emerged with strong performance. Here, we review current advances of computer vision identification techniques to provide both computer scientists and biologists with an overview of the available tools and discuss their applications. We conclude by offering recommendations for starting an animal identification project, illustrate current limitations, and propose how they might be addressed in the future.

Controlled experiments to explore the use of a multi-tissue approach to characterizing stress in wild-caught Pacific halibut (Hippoglossus stenolepis).

The integration of multiple tissues in physiological and ecological analyses can enhance methodological approaches, increase applications for data and extend interpretation of results. Previous investigations of the stress response in fish have focused primarily on cortisol levels in a single matrix-blood plasma-which confines interpretations of cortisol levels to a short temporal frame. Epidermal mucus has been proposed as an alternative or complement to plasma that may provide a view to cortisol levels over a different temporal window allowing comparative assessment. Here, we explore the potential for multi-tissue cortisol analysis using both plasma and epidermal mucus in Pacific halibut (Hippoglossus stenolepis). The relative timing at which cortisol increased and decreased in the two matrices as well as cortisol concentrations at estimated peak levels were compared in two trials after (i) inducing cortisol synthesis by adrenocorticotropic hormone (ACTH1-24) administration and (ii) inducing cortisol elimination using cortisol (hydrocortisone, 98%) injection. The ACTH treatment elicited a peak plasma cortisol response approximately 12 hours post-injection, while mucus cortisol concentrations peaked later at approximately 62 hours post-injection. Exogenous cortisol treatments suggested relatively little transfer of cortisol from plasma to mucus, potentially reflecting differential effects of endogenous and exogenous cortisol. Our results suggest the potential utility of mucus as a sampling matrix that provides an extended window for detection of the stress response as compared to plasma. Results also suggest the utility of a multi-tissue approach to cortisol analysis with potential applications to applied fisheries research. Increased understanding of the relative scale of the cortisol response to stress (e.g. capture) will allow researchers and managers to better interpret the physiological condition and survival outcome of fish subjected to regulatory discard.

Gene Co-Expression Network Analysis Identifies Vitamin D-Associated Gene Modules in Adult Normal Rectal Epithelium Following Supplementation.

Colorectal cancer (CRC) is a common, multifactorial disease. While observational studies have identified an association between lower vitamin D and higher CRC risk, supplementation trials have been inconclusive and the mechanisms by which vitamin D may modulate CRC risk are not well understood. We sought to perform a weighted gene co-expression network analysis (WGCNA) to identify modules present after vitamin D supplementation (when plasma vitamin D level was sufficient) which were absent before supplementation, and then to identify influential genes in those modules. The transcriptome from normal rectal mucosa biopsies of 49 individuals free from CRC were assessed before and after 12 weeks of 3200IU/day vitamin D (Fultium-D3) supplementation using paired-end total RNAseq. While the effects on expression patterns following vitamin D supplementation were subtle, WGCNA identified highly correlated genes forming gene modules. Four of the 17 modules identified in the post-vitamin D network were not preserved in the pre-vitamin D network, shedding new light on the biochemical impact of supplementation. These modules were enriched for GO terms related to the immune system, hormone metabolism, cell growth and RNA metabolism. Across the four treatment-associated modules, 51 hub genes were identified, with enrichment of 40 different transcription factor motifs in promoter regions of those genes, including VDR:RXR. Six of the hub genes were nominally differentially expressed in studies of vitamin D effects on adult normal mucosa organoids: LCN2, HLA-C, AIF1L, PTPRU, PDE4B and IFI6. By taking a gene-correlation network approach, we have described vitamin D induced changes to gene modules in normal human rectal epithelium in vivo, the target tissue from which CRC develops.

Quantifying and addressing the prevalence and bias of study designs in the environmental and social sciences.

Building trust in science and evidence-based decision-making depends heavily on the credibility of studies and their findings. Researchers employ many different study designs that vary in their risk of bias to evaluate the true effect of interventions or impacts. Here, we empirically quantify, on a large scale, the prevalence of different study designs and the magnitude of bias in their estimates. Randomised designs and controlled observational designs with pre-intervention sampling were used by just 23% of intervention studies in biodiversity conservation, and 36% of intervention studies in social science. We demonstrate, through pairwise within-study comparisons across 49 environmental datasets, that these types of designs usually give less biased estimates than simpler observational designs. We propose a model-based approach to combine study estimates that may suffer from different levels of study design bias, discuss the implications for evidence synthesis, and how to facilitate the use of more credible study designs.

A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis.

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group-synchronous ovarian developmental pattern with determinate fecundity and support for their batch-spawning strategy.

Development and simulation of fully glycosylated molecular models of ACE2-Fc fusion proteins and their interaction with the SARS-CoV-2 spike protein binding domain.

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.

New Means to Control Molecular Assembly.

While self-assembly of molecules is relatively well-known and frequently utilized in chemical synthesis and material science, controlled assembly of molecules represents a new concept and approach. The present work demonstrates the concept of controlled molecular assembly using a non-spherical biomolecule, heparosan tetrasaccharide (MW = 1.099 kD). The key to controlled assembly is the fact that ultra-small solution droplets exhibit different evaporation dynamics from those of larger ones. Using an independently controlled microfluidic probe in an atomic force microscope, sub-femtoliter aqueous droplets containing designed molecules produce well-defined features with dimensions as small as tens of nanometers. The initial shape of the droplet and the concentration of solute within the droplet dictate the final assembly of molecules due to the ultrafast evaporation rate and dynamic spatial confinement of the droplets. The level of control demonstrated in this work brings us closer to programmable synthesis for chemistry and materials science which can be used to develop vehicles for drug delivery three-dimensional nanoprinting in additive manufacturing.

Label-free spectroscopic tissue characterization using fluorescence excitation-scanning spectral imaging.

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation-scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation-scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi-pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure-specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation-scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation-scanning technologies to clinical imaging platforms.

Exploring sample cross-contamination in fish epidermal mucus.

Cross-contamination of epidermal mucus was assessed at three sampling locations on the bodies of Pacific halibut Hippoglossus stenolepis by inducing contact between fish coated with labelled synthetic mucus and non-treated fish. Results indicate a positive relationship between sampling site exposure and sample contamination and that mucous sample cross-contamination can be mitigated by sampling in a location protected from external contact.