Mining electron density for functionally relevant protein polysterism in crystal structures.

This review focuses on conceptual and methodological advances in our understanding and characterization of the conformational heterogeneity of proteins. Focusing on X-ray crystallography, we describe how polysterism, the interconversion of pre-existing conformational substates, has traditionally been analyzed by comparing independent crystal structures or multiple chains within a single crystal asymmetric unit. In contrast, recent studies have focused on mining electron density maps to reveal previously 'hidden' minor conformational substates. Functional tests of the importance of minor states suggest that evolutionary selection shapes the entire conformational landscape, including uniquely configured conformational substates, the relative distribution of these substates, and the speed at which the protein can interconvert between them. An increased focus on polysterism may shape the way protein structure and function is studied in the coming years

Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.Australian National University PhD Scholarship Award to Sunita Biswas

Kinetic and sequence-structure-function analysis of known LinA variants with different hexachlorocyclohexane isomers

BACKGROUND Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (-)-α -HCH are also discussed. METHODOLOGY/PRINCIPAL FINDINGS Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer. CONCLUSIONS/SIGNIFICANCE The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.This work was supported by the Indo-Australian Biotechnology Fund from the Department of Education Science and Technology (DEST), Australia and the Department of Biotechnology (DBT), India

Unintended and accidental medical radiation exposures in radiology: guidelines on investigation and prevention

This paper sets out guidelines for managing radiation exposure incidents involving patients in diagnostic and interventional radiology. The work is based on collation of experiences from representatives of international and national organizations for radiologists, medical physicists, radiographers, regulators, and equipment manufacturers, derived from an International Atomic Energy Agency Technical Meeting. More serious overexposures can result in skin doses high enough to produce tissue reactions, in interventional procedures and computed tomography, most notably from perfusion studies. A major factor involved has been deficiencies in training of staff in operation of equipment and optimization techniques. The use of checklists and time outs before procedures commence, and dose alerts when critical levels are reached during procedures can provide safeguards to reduce risks of these effects occurring. However, unintended and accidental overexposures resulting in relatively small additional doses can take place in any diagnostic or interventional X-ray procedure and it is important to learn from errors that occur, as these may lead to increased risks of stochastic effects. Such events may involve the wrong examinations, procedural errors, or equipment faults. Guidance is given on prevention, investigation and dose calculation for radiology exposure incidents within healthcare facilities. Responsibilities should be clearly set out in formal policies, and procedures should be in place to ensure that root causes are identified and deficiencies addressed. When an overexposure of a patient or an unintended exposure of a foetus occurs, the foetal, organ, skin and/or effective dose may be estimated from exposure data. When doses are very low, generic values for the examination may be sufficient, but a full assessment of doses to all exposed organs and tissues may sometimes be required. The use of general terminology to describe risks from stochastic effects is recommended rather than calculation of numerical values, as these are misleading when applied to individuals

Understanding enzymes specificities as a tool for cofactor engineering

S-adenosylmethionine (SAM) is the second most abundant cofactor in nature after adenosyl triphosphate (ATP). SAM is synthesized in a cell by Methionine adenosyltransferase (MAT) using ATP and methionine and it is used as a methyl donor to methylate different substrates (DNA, protein, RNA, small molecules) by methyltransferases (Mtases). The methylation is a key process for cellular regulation and aberrant methylation associated with the disease condition. We are aiming at engineering two-steps pathway for an orthogonal cofactor, for this purpose we first decided to explore the promiscuities of the nucleotide base of ATP for two enzymes (MAT and Mtases). To find good candidates for these engineering we expressed and purified MAT from different organisms. We found MAT from specific organisms are promiscuous for the new nucleotide-based cofactor. It is very interesting that certain MAT is promiscuous for nucleotides-based cofactor but are these newly formed cofactors also promiscuous for the methyltransferase (Mtases) who is the main user of theses cofactors? Further, we have also investigated promiscuity of the DNA methyltransferase (Mtases) from bacteria. Overall these findings lay the foundation for our engineering studies and hint at the evolution of these enzymes. References: Gade, M., Villar-Briones, A.; Laurino, P. “Understanding Enzymes Promiscuity for Novel Cofactors” Submitted. G. L. Cantoni, J. Am. Chem. Soc. 1952, 74, 2942-2943. O. Khersonsky, D. S. Tawfik, Annu Rev Biochem 2010, 79, 471-505

Tree thinning and fire affect ectomycorrhizal fungal communities and enzyme activities

Common ecological restoration treatments such as thinning trees and prescribed burning could result in changes to soil fungal communities and changes to the function of those communities. Ectomycorrhizal fungi are especially likely to be affected as they are symbionts on plant roots and exhibit host and niche preferences. Ectomycorrhizal fungi also produce extracellular enzymes that are important in soil nutrient cycling. We conducted a community survey of ectomycorrhizal fungi and assayed ectomycorrhizal root tip enzyme activity using substrate plugs in northern Mississippi upland oak–pine woodland plots differing in restoration history to explore the influence of woodland restoration on ectomycorrhizalfungal community composition and function. Restoration treatment was significant in explaining the occurrence of the most common fungal species (Russula xerampelina) and the most common family (Thelephoraceae) in the ectomycorrhizal fungal community survey. Highest potential laccase, peroxidase, and N-acetyl-b-D-glucosaminidase enzyme activity were found in a prescribed burn plot, and the lowest enzyme activities at a wildfire plot, where richness of ectomycorrhizal fungi was also lower. Different fungal families displayed significantly different enzymatic capabilities, with Hydnangiaceae having the highest laccase activity and Tuberaceae having significantly higher peroxidase and chitinase activity than several other families. These results suggest that restoration treatments can affect ectomycorrhizal fungal community composition and function, and better understanding these changes can aid understanding of the niches of ectomycorrhizal fungi and the impacts of restoration

Timescales of variation in diversity and production of bacterioplankton assemblages in the Lower Mississippi River

Copyright: © 2020 Payne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Rivers are characterized by rapid and continuous one-way directional fluxes of flowing, aqueous habitat, chemicals, suspended particles, and resident plankton. Therefore, at any particular location in such systems there is the potential for continuous, and possibly abrupt, changes in diversity and metabolic activities of suspended biota. As microorganisms are the principal catalysts of organic matter degradation and nutrient cycling in rivers, examination of their assemblage dynamics is fundamental to understanding system-level biogeochemical patterns and processes. However, there is little known of the dynamics of microbial assemblage composition or production of large rivers along a time interval gradient. We quantified variation in alpha and beta diversity and production of particle-associated and free-living bacterioplankton assemblages collected at a single site on the Lower Mississippi River (LMR), the final segment of the largest river system in North America. Samples were collected at timescales ranging from days to weeks to months up to a year. For both alpha and beta diversity, there were similar patterns of temporal variation in particle-associated and free-living assemblages. Alpha diversity, while always higher on particles, varied as much at a daily as at a monthly timescale. Beta diversity, in contrast, gradually increased with time interval of sampling, peaking between samples collected 180 days apart, before gradually declining between samples collected up to one year apart. The primary environmental driver of the temporal pattern in beta diversity was temperature, followed by dissolved nitrogen and chlorophyll a concentrations. Particle-associated bacterial production corresponded strongly to temperature, while free-living production was much lower and constant over time. We conclude that particle-associated and free-living bacterioplankton assemblages of the LMR vary in richness, composition, and production at distinct timescales in response to differing sets of environmental factors. This is the first temporal longitudinal study of microbial assemblage structure and dynamics in the LMR

Three-dimensional reconstruction of autologous vein bypass graft distal anastomoses imaged with magnetic resonance: clinical and research applications

AbstractHigh-resolution magnetic resonance imaging was combined with computational modeling to create focused three-dimensional reconstructions of the distal anastomotic region of autologous vein peripheral bypass grafts in a preliminary series of patients. Readily viewed on a personal computer or printed as hard copies, a detailed appreciation of in vivo postoperative features of the anastomosis is possible. These reconstructions are suitable for analysis of geometric features, including vessel caliber, tortuosity, anastomotic angles, and planarity. Some potential clinical and research applications of this technique are discussed

Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible

Three-dimensional reconstruction of autologous vein bypass graft distal anastomoses imaged with magnetic resonance: clinical and research applications

AbstractHigh-resolution magnetic resonance imaging was combined with computational modeling to create focused three-dimensional reconstructions of the distal anastomotic region of autologous vein peripheral bypass grafts in a preliminary series of patients. Readily viewed on a personal computer or printed as hard copies, a detailed appreciation of in vivo postoperative features of the anastomosis is possible. These reconstructions are suitable for analysis of geometric features, including vessel caliber, tortuosity, anastomotic angles, and planarity. Some potential clinical and research applications of this technique are discussed