Intra-molecular lysine-arginine derived advanced glycation end-product cross-linking in Type I collagen: A molecular dynamics simulation study

Covalently cross-linked advanced glycation end products (AGE) are among the major post-translational modifications to proteins as a result of non-enzymatic glycation. The formation of AGEs has been shown to have adverse effects on the properties of the collagenous tissue; they are even linked to a number of age related disorders. Little is known about the sites at which these AGEs form or why certain sites within the collagen are energetically more favourable than others. In this study we have used a proven fully atomistic molecular dynamics approach to identify six sites where the formation of the intra-molecular 3-deoxyglucosone-derived imidazolium cross-link (DOGDIC) is energetically favourable. We have also conducted a comparison of these positions with those of the more abundant glucosepane cross-link, to determine any site preference. We show that when we consider both lysine and arginine AGEs, they exhibit a prevalence to form within the gap region of the collagen fibril

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule’s size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules

Effect on the mechanical properties of type I collagen of intra-molecular lysine-arginine derived advanced glycation end-product cross-linking

Non-enzymatic advanced glycation end product (AGE) cross-linking of collagen molecules has been hypothesised to result in significant changes to the mechanical properties of the connective tissues within the body, potentially resulting in a number of age related diseases. We have investigated the effect of two of these cross-links, glucosepane and DOGDIC, on the tensile and lateral moduli of the collagen molecule through the use of a steered molecular dynamics approach, using previously identified preferential formation sites for intra-molecular cross-links. Our results show that the presence of intra-molecular AGE cross-links increases the tensile and lateral Young’s moduli in the low strain domain by between 3.0–8.5% and 2.9–60.3% respectively, with little effect exhibited at higher strains

The Value of Participating in British Exploring Society Expeditions: A Three Year Multi Cohort Study

A primary aim of many expeditions is to facilitate personal development of young people and 15 while there is much anecdotal evidence to support this aim, there is limited empirical work of 16 varied quality that explores the specific nature of such benefits. This research examined nine 17 summer BES expeditions (Norway, Namibia & Amazon in 2012; Finnmark, Ladakh and 18 Namibia in 2013 and 2014) involving 58 young people (aged between 15 and 22) who completed 19 three on-line questionnaires to collect qualitative (open ended questions) and quantitative (likert 20 scale) data. Measurement of four psychological attributes associated with effective character 21 development were used: mental toughness, coping skills, GRIT and leadership skills. Surveys 22 were completed at three stages; 1) pre expedition, 2) immediately post expedition and 3) three 23 months post expedition. Results indicated that the expeditions impacted positively on the 24 psychological attributes of young people, with short term effects (three months). 25 For the 58 participants, there were statistically significant improvements and small positive 26 effect sizes in mental toughness (P=.006; ηp 2 .167), leadership skills (P=.004; ηp 2 .18), and 27 GRIT (P=.001; ηp 2 .218). There was no significant difference (P>0.05) or effect size for the 28 application of coping strategies. Qualitative data provided insights into how the learning took 29 place and individual perspectives on the overall value of the expedition in relation to narrative 30 understandin

Mechanical Polyprotein Assembly Using Sfp and Sortase‐Mediated Domain Oligomerization for Single‐Molecule Studies

Single‐molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) provides remarkable details on the energy landscapes governing protein (un)folding and intermolecular complex dissociation. In such experiments, multidomain polyproteins consisting of multiple copies of independently foldable domains provide internal controls identifiable by characteristic contour length increments, unfolding forces, and/or unfolding substeps. Here, a new approach to polyprotein synthesis is presented relying on posttranslational enzyme‐mediated oligomerization of domains. Mutant variants of immunoglobulin 27 (I27) and a bacterial cellulose binding module (CBM) fused to an Ig‐like X‐module (XMod), and a mechanostable receptor called Dockerin (Doc) are produced with complementary peptide tags. By utilizing 4′‐phosphopantetheinyl transferase and Sortase A, the system enables I27‐domain oligomerization into polyproteins of varying lengths followed by C‐terminal capping with mechanostable Doc. The number of oligomerized domains per molecule, the unfolding forces, and the complex rupture forces of posttranslationally assembled polyproteins are characterized using >40 h automated AFM–SMFS with a Cohesin (Coh)‐modified cantilever. Use of the Coh–Doc interaction to unfold polyproteins provides a high yield of ≈ 3800 specific single‐molecule interaction curves. This approach is advantageous for assembly of polyproteins from domains that lack proper folding or are insoluble in a polyprotein format

Enzyme-mediated hydrogel encapsulation of single cells for high-throughput screening and directed evolution of oxidoreductases

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol cross-coupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H; 2; O; 2; -producing enzymes many folds

Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4`-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects

Genetically encoded stimuli-responsive cytoprotective hydrogel capsules for single cells provide novel genotype-phenotype linkage

Modification of cell surfaces with synthetic polymers is a promising approach for regulating cellular behavior. Here, we describe a genetically controlled strategy for selectively encapsulating single yeast cells in synthetic microniches comprising cross-linked phenol-modified alginate and chitosan hydrogel capsules. Our system links inducible gene expression with enzyme-mediated hydrogel polymerization and provides a novel genotype–phenotype linkage whereby only cells carrying a requisite gene encoding a flavin adenine dinucleotide-dependent oxidoreductase undergo autonomous enzyme-mediated surface polymerization, resulting in the formation of hydrogel capsules. The composition of the hydrogel capsules is highly tunable and the capsule sizes are pH-responsive, allowing for control of capsule porosity and shell diameters over a range of 15–80 μm. The hydrogel capsules prevent extracellular proteins from reaching the cell surface, thereby conferring cellular immunity to lytic enzyme cocktails and rendering the hydrogel capsules cytoprotective against osmotic shock. We demonstrate the utility of this genetically controlled artificial hydrogel-encapsulated cell phenotype by isolating and enriching uniform eukaryotic cell lineages from genetically heterogeneous cell mixtures at 95–100% efficiency. The encapsulated cells remained viable and were capable of dividing and breaking free from their hydrogel capsules, allowing further propagation of selected cells. Our bottom-up approach to cellular compartmentalization links inducible intracellular genetic components with an artificial extracellular matrix that resists enzymatic lysis and mediates communication with the surrounding environment through a size-tunable and permeable hydrogel capsule

Conservation genomics reveals possible illegal trade routes and admixture across pangolin lineages in Southeast Asia

The use of genome-wide genetic markers is an emerging approach for informing evidence-based management decisions for highly threatened species. Pangolins are the most heavily trafficked mammals across illegal wildlife trade globally, but critically endangered Sunda pangolins (Manis javanica) have not been widely studied in insular Southeast Asia. We used > 12,000 single nucleotide polymorphic markers (SNPs) to assign pangolin seizures from illegal trade of unknown origin to possible geographic sources via genetic clustering with pangolins of known origin. Our SNPs reveal three previously unrecognized genetic lineages of Sunda pangolins, possibly from Borneo, Java and Singapore/Sumatra. The seizure assignments suggest the majority of pangolins were traded from Borneo to Java. Using mitochondrial markers did not provide the same resolution of pangolin lineages, and to explore if admixture might explain these differences, we applied sophisticated tests of introgression using > 2000 SNPs to investigate secondary gene flow between each of the three Sunda pangolin lineages. It is possible the admixture which we discovered is due to human-mediated movements of pangolins. Our findings impact a range of conservation actions, including tracing patterns of trade, repatriation of rescue animals, and conservation breeding. In order to conserve genetic diversity, we suggest that, pending further research, each pangolin lineage should as a precaution be protected and managed as an evolutionarily distinct conservation unit

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS