Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods

The structure of α-crustacyanin has been determined to 30 Å resolution using negative-stain electron microscopy (EM) single-particle averaging and modelling with the β-crustacyanin dimer from the crystal structure (Protein Data Bank code 1gka), guided by PISA protein subunit interface calculations for 1gka, and compared with the protein arrangements observed in the crystal lattice of 1gka. This α-crustacyanin EM model has been checked against SAXS experimental data, including comparison with rigid-body models calculated from the SAXS data, and finally with analytical ultracentrifugation measurements

Interaction standards for biophysics: anti-lysozyme nanobodies

From Springer Nature via Jisc Publications RouterHistory: received 2020-10-14, rev-recd 2021-03-01, registration 2021-03-22, accepted 2021-03-22, pub-electronic 2021-04-11, online 2021-04-11, pub-print 2021-05Publication status: PublishedFunder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100004440; Grant(s): 203128/Z/16/ZAbstract: There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb’s or ‘nanobodies’ against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards

The lexicon of antimicrobial peptides: a complete set of arginine and tryptophan sequences

Our understanding of the activity of cationic antimicrobial peptides (AMPs) has focused on well-characterized natural sequences, or limited sets of synthetic peptides designed de novo. We have undertaken a comprehensive investigation of the underlying primary structural features that give rise to the development of activity in AMPs. We consider a complete set of all possible peptides, up to 7 residues long, composed of positively charged arginine (R) and / or hydrophobic tryptophan (W), two features most commonly associated with activity. We found the shortest active peptides were 4 or 5 residues in length, and the overall landscapes of activity against gram-positive and gram-negative bacteria and a yeast were positively correlated. For all three organisms we found a single activity peak corresponding to sequences with around 40% R; the presence of adjacent W duplets and triplets also conferred greater activity. The mechanistic basis of these activities comprises a combination of lipid binding, particularly to negatively charged membranes, and additionally peptide aggregation, a mode of action previously uninvestigated for such peptides. The maximum specific antimicrobial activity appeared to occur in peptides of around 10 residues, suggesting ‘diminishing returns’ for developing larger peptides, when activity is considered per residue of peptide

Structural basis for complement factor H-linked age-related macular degeneration

This is the final version of the article. Available from the publisher via the DOI in this record.Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.B. Prosser is funded by the Wellcome Trust Structural Biology Training Program (075415/Z/04/Z). S. Johnson and P. Roversi were funded by grants to S.M. Lea from the Medical Research Council (MRC) of the United Kingdom (grants G0400389 and G0400775). D. Uhrin and P.N. Barlow were funded by the Wellcome Trust (078780/ Z/05/Z). S.J. Clark was funded by an MRC Doctoral Training Account (G78/7925), and R.B. Sim and A.J. Day were funded by MRC core funding to the MRC Immunochemistry Unit

Variability and physical demands of international seam bowlers in one-day and Twenty20 international matches across five years

Objectives: To quantify and compare the match demands and variability of international One4 Day (ODI) with Twenty20 (T20) cricket matches and to compare ODI match demands when5 competing home and away. Design: Single cohort, longitudinal observation. Methods: Thirteen international male seam bowlers across 204 matches (ODI= 160; T20= 44) were investigated over five-years (2015-2019). Using global positioning sensors and accelerometers, physical demands were quantified using distance covered at different velocities and the number of entries into high and low intensity acceleration and deceleration bands. Variability was quantified using coefficient of variation (CV) and smallest worthwhile change. Results: Significantly greater (p4 m∙s2 19 (within-player ODI CV= 79.2%. T20 CV= 77.2%. Between-player ODI CV= 84.7%. T20= 38.8%) and distance covered >25 km∙h-1 20 (within-player ODI CV = 65.5%. T20= 64.1%) showed the greatest variability. Conclusions: Players are exposed to different physical demands in ODI Vs T20 matches, but not for home Vs away ODI matches. Practitioners should be aware of the large variability in high-speed/intensity accelerations and decelerations across matches

No association between intravenous fluid volume and endothelial glycocalyx shedding in patients undergoing resuscitation for sepsis in the emergency department

Endothelial glycocalyx (EG) shedding is associated with septic shock and described following intravenous (IV) fluid administration. To investigate the possible impact of IV fluids on the pathobiology of septic shock we investigated associations between biomarkers of EG shedding and endothelial cell activation, and relationships with IV fluid volume. Serum samples were obtained on admission (T0) and at 24 h (T24) in patients undergoing haemodynamic resuscitation for suspected septic shock in the emergency department. Biomarkers of EG shedding—Syndecan-1 (Syn-1), Syndecan-4 (Syn-4), Hyaluronan, endothelial activation—Endothelin-1 (ET-1), Angiopoeitin-2 (Ang-2), Vascular Endothelial Growth Factor Receptor-1(VEGF-1) and leucocyte activation/inflammation—Resistin, Neutrophil Gelatinase Associated Lipocalin (NGAL) and a marker of cardiac stretch—Pro-Atrial Natriuretic Peptide (Pro-ANP) were compared to the total IV fluid volume administered using Tobit regression. Data on 86 patients (52 male) with a mean age of 60 (SD 18) years were included. The mean fluid volume administered to T24 was 4038 ml (SD 2507 ml). No significant association between fluid volume and Pro-ANP or any of the biomarkers were observed. Syn-1 and Syn-4 were significantly correlated with each other (Spearman Rho 0.43, p \u3c 0.001) but not with Hyaluronan. Syn-1 and Syn-4 both correlated with VEGFR-1 (Rho 0.56 and 0.57 respectively, p \u3c 0.001) whereas Hyaluronan correlated with ET-1 (Rho 0.43, p \u3c 0.001) and Ang-2 (Rho 0.43, p \u3c 0.001). There was no correlation between Pro-ANP and any of the EG biomarkers. Distinct patterns of association between biomarkers of EG shedding and endothelial cell activation were observed among patients undergoing resuscitation for sepsis. No relationship between IV fluid volume and Pro-ANP or any of the other biomarkers was observed

The lexicon of antimicrobial peptides: a complete set of arginine and tryptophan sequences

From Springer Nature via Jisc Publications RouterHistory: received 2020-08-09, accepted 2021-03-29, registration 2021-04-24, pub-electronic 2021-05-21, online 2021-05-21, collection 2021-12Publication status: PublishedFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/M011208/1Abstract: Our understanding of the activity of cationic antimicrobial peptides (AMPs) has focused on well-characterized natural sequences, or limited sets of synthetic peptides designed de novo. We have undertaken a comprehensive investigation of the underlying primary structural features that give rise to the development of activity in AMPs. We consider a complete set of all possible peptides, up to 7 residues long, composed of positively charged arginine (R) and / or hydrophobic tryptophan (W), two features most commonly associated with activity. We found the shortest active peptides were 4 or 5 residues in length, and the overall landscapes of activity against gram-positive and gram-negative bacteria and a yeast were positively correlated. For all three organisms we found a single activity peak corresponding to sequences with around 40% R; the presence of adjacent W duplets and triplets also conferred greater activity. The mechanistic basis of these activities comprises a combination of lipid binding, particularly to negatively charged membranes, and additionally peptide aggregation, a mode of action previously uninvestigated for such peptides. The maximum specific antimicrobial activity appeared to occur in peptides of around 10 residues, suggesting ‘diminishing returns’ for developing larger peptides, when activity is considered per residue of peptide

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins.

Funder: AstraZenecaFunder: AstraZeneca postdoc fundBromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics

Variability and physical demands of international seam bowlers in one-day and Twenty20 international matches across five years.

Objectives: To quantify and compare the match demands and variability of international One-Day (ODI) with Twenty20 (T20) cricket matches and to compare ODI match demands when competing home and away. Design: Single cohort, longitudinal observation. Methods: Thirteen international male seam bowlers across 204 matches (ODI= 160; T20= 44) were investigated over five-years (2015-2019). Using global positioning sensors and accelerometers, physical demands were quantified using distance covered at different velocities and the number of entries into high and low intensity acceleration and deceleration bands. Variability was quantified using coefficient of variation (CV) and smallest worthwhile change. Results: Significantly greater (p4 m∙s2 (within-player ODI CV= 79.2%. T20 CV= 77.2%. Between-player ODI CV= 84.7%. T20= 38.8%) and distance covered >25 km∙h-1 (within-player ODI CV= 65.5%. T20= 64.1%) showed the greatest variability. Conclusions: Players are exposed to different physical demands in ODI Vs T20 matches, but not for home Vs away ODI matches. Practitioners should be aware of the large variability in high-speed/intensity accelerations and decelerations across matches

Variability of test match cricket and the effects of match location on physical demands in male seam bowlers

The physical demands of test match cricket in seam bowlers during fielding are currently unknown. Similarly, analysis of between-match variability and the effects of playing home vs. away is required. Nine international male seam bowlers across 28 test matches (n= 9 home; n= 19 away) were investigated over five years (2015-2019). Seam bowlers wore global positioning sensors during match play fielding to quantify physical demands. Absolute and relative (per hour) distances covered in five velocity bands, total distance, and number of accelerations and decelerations were assessed for each match. Coefficient of variation (CV%) and smallest worthwhile change were used to calculate between-match variability. Mixed linear modelling was used to analyse home vs away matches. Seam bowlers covered up to 50 km, with maximal durations of >21 hours during test match fielding. Small between-match CV% (8.3) were found for maximal velocity with large (CV% = 21-192) between-match variability across most other variables. Greater distances were covered at 15-20 km·h-1 (p= 0.02) and >25 km·h-1 (p= 0.04) when playing at home. The results demonstrated substantial, highly variable physical demands. Practitioners should adapt training retrospectively to the match demands encountered and should anticipate that match intensity may be higher during home matches