Insights into enzymatic halogenation from computational studies

The halogenases are a group of enzymes that have only come to the fore over the last 10 years thanks to the discovery and characterization of several novel representatives. They have revealed the fascinating variety of distinct chemical mechanisms that nature utilizes to activate halogens and introduce them into organic substrates. Computational studies using a range of approaches have already elucidated many details of the mechanisms of these enzymes, often in synergistic combination with experiment. This Review summarizes the main insights gained from these studies. It also seeks to identify open questions that are amenable to computational investigations. The studies discussed herein serve to illustrate some of the limitations of the current computational approaches and the challenges encountered in computational mechanistic enzymology

Amino acid sequence, haem-iron co-ordination geometry and functional properties of mitochondrial and bacterial c-type cytochromes

Cytochromes are found in all biological oxidation Systems which involve transport of reducing equivalents through organized chains of membrane bound intermediates, regardless of the ultimate oxidant (Keilin, 1966; Bartsch, 1978; Meyer & Kamen, 1982). Thus, cytochromes are present not only in the aerobic mitochondrial and bac-terial respiratory chain, but are also found in much more diversified procariotic Systems, including all varieties of facultative anaerobes (nitrate and nitrite reducers), obligate anaerobes (sulphate reducers and phototrophic sulphur bacteria), facultative photoheterotrophes (phototrophic non-sulphur purple bacteria), and the photoautotrophic cyanobacteria (blue-green algae). Among the different types of cytochromes occurring in the cell, the soluble c-type cytochromes (‘class I', Meyer & Kamen, 1982) are the most abundant and best characterized group of proteins (Bartsch, 1978; Meyer & Kamen, 1982; Dickerson & Timkovitch, 1975; Lemberg & Barrett, 1973; Salemme, 1977; Ferguson-Miller, Brautigan & Margoliash, 1979). The amino acid sequences of more than 80 mitochrondrial and close to 40 bacterial cytochromes c are known (Meyer & Kamen, 1982; Dickerson & Timkovitch, 1975; Schwartz & Dayhoff, 1976; Dayhoff & Barker, 1978

An investigation into the unusual linkage isomerization and nitrite reduction activity of a novel tris(2-pyridyl) copper complex

The copper-containing nitrite reductases (CuNIRs) are a class of enzymes that mediate the reduction of nitrite to nitric oxide in biological systems. Metal–ligand complexes that reproduce the salient features of the active site of CuNIRs are therefore of fundamental interest, both for elucidating the possible mode of action of the enzymes and for developing biomimetic catalysts for nitrite reduction. Herein, we describe the synthesis and characterization of a new tris(2-pyridyl) copper complex ([Cu1(NO2)2]) that binds two molecules of nitrite, and displays all three of the common binding modes for NO2−, with one nitrite bound in an asymmetric quasi-bidentate κ2-ONO manner and the other bound in a monodentate fashion with a linkage isomerism between the κ1-ONO and κ1-NO2 binding modes. We use density functional theory to help rationalize the presence of all three of these linkage isomers in one compound, before assessing the redox activity of [Cu1(NO2)2]. These latter studies show that the complex is not a competent nitrite reduction electrocatalyst in non-aqueous solvent, even in the presence of additional proton donors, a finding which may have implications for the design of biomimetic catalysts for nitrite reduction

Top-down Dendritic Input Increases the Gain of Layer 5 Pyramidal Neurons

The cerebral cortex is organized so that an important component of feedback input from higher to lower cortical areas arrives at the distal apical tufts of pyramidal neurons. Yet, distal inputs are predicted to have much less impact on firing than proximal inputs. Here we show that even weak asynchronous dendritic input to the distal tuft region can significantly increase the gain of layer 5 pyramidal neurons and thereby the output of columns in the primary somatosensory cortex of the rat. Noisy currents injected in ramps at different dendritic locations showed that the initial slope of the frequency-current (f/I) relationship increases with the distance of the current injection from the soma. The increase was due to the interaction of dendritic depolarization with back-propagating APs which activated dendritic calcium conductances. Gain increases were accompanied by a change of firing mode from isolated spikes to bursting where the timing of bursts coded the presence of coincident somatic and dendritic inputs. We propose that this dendritic gain modulation and the timing of bursts may serve to associate top-down and bottom-up input on different time scale

[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13Cα evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time perio

Supporting students in the transition to postgraduate taught study in STEM subjects

While there has been a wide range of studies examining the transition of undergraduate and postgraduate research students, there are few which concentrate on the experiences of postgraduate taught (PGT) students. This is unfortunate, because PGT students have pressing needs for support: since taught masters courses last for usually one academic year, postgraduate students are asked to adapt and succeed at a far faster rate than undergraduates, who take four years in Scotland to complete an honours degree. PGT students are a minority group amongst the university population, with e.g. more than three times as many undergraduates enrolled at the University of Glasgow than postgraduates. Furthermore, international students represent a high proportion of PGT students. To better understand the needs of PGT students and therefore improve the quality of their education, we need to understand their experiences and challenges as they transition through their course. This paper presents a study focused on PGT students in STEM subjects at the University of Glasgow. Feedback from students in the College of Science and Engineering was gathered using a multi-methodological approach. Surveys, one-to-one interviews and a workshop were utilised to investigate students’ perceptions of support received from staff and services. This data was linked to student academic confidence, social confidence, and overall satisfaction with their experience at the university. Data were gathered at three points in the year to evaluate whether perceptions change as students progress through their course. This data from surveys and interviews was used to direct a workshop, which discussed potential solutions to issues raised. Better online resources were identified as key to feeling prepared before the commencement of a PGT course, and better communication with lecturers and peers was important to the success and satisfaction of students, particularly after beginning PGT study

Evaluating genetic traceability methods for captive bred marine fish and their applications in fisheries management and wildlife forensics

Growing demands for marine fish products is leading to increased pressure on already depleted wild populations and a rise in the aquaculture production. Consequently, more captive bred fish are released into the wild through accidental escape or deliberate restocking, stock enhancement and sea ranching programs. The increased mixing of captive bred fish with wild conspecifics may affect the ecological and/or genetic integrity of wild fish populations. From a fisheries management perspective unambiguous identification tools for captive bred fish will be highly valuable to manage risks. Additionally there is great potential to use these tools in wildlife forensics (i.e. tracing back escapees to their origin and determining mislabelling of seafood products). Using SNP data from captive bred and wild populations of Atlantic cod (Gadus morhua L.) and sole (Solea solea L.), we explored the efficiency of population and parentage assignment techniques for the identification and tracing of captive bred fish. Simulated and empirical data were used to correct for stochastic genetic effects. Overall, parentage assignment performed well when a large effective population size characterizes the broodstock and escapees originate from early generations of captive breeding. Consequently, parentage assignments are particularly useful from a fisheries management perspective to monitor the effects of deliberate releases of captive bred fish on wild populations. Population assignment proved to be more efficient after several generations of captive breeding, which makes it a useful method in forensic applications for well-established aquaculture species. We suggest the implementation of a case by case strategy when choosing the best method

MitoNeoD:a mitochondria-targeted superoxide probe

Mitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅−production in health and disease