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Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. "Crab"-like configuration of two catalytic peptides ("pincers") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for " single-pincer" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled "Crabs" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5′-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Intervention fidelity in health services research has been poor with a reported lack of understanding about what constitutes pragmatic adaptation of interventions and what constitutes failure to maintain intervention fidelity. However, the challenges facing those delivering such interventions have not been thoroughly explored. The aims of this study were to critically explore the challenges in maintaining fidelity experienced by physiotherapy staff and support workers when delivering a complex intervention for older people living with frailty. Methods: This study is a secondary analysis of data from a process evaluation of a large randomised controlled trial (RCT). The process evaluation employed qualitative methodologies with mixed methods including a variety of data collection methods, including participant observation, semi-structured interviews and documentary analysis. Thematic analysis was used to make sense of the data. Results: Many therapy staff felt ongoing confusion about what was acceptable to adapt and what needed to follow the protocol exactly. We found that some therapy staff were able to embrace the challenges of pragmatically adapting interventions while maintaining intervention fidelity, others stuck rigidly to the protocol and failed to adapt interventions where it was necessary. Conclusion: It was clear that the understanding of fidelity and pragmatism was poor. While pragmatic trials are vital to replicate real world clinical practice, further guidance may need to be developed in order to guide the level of adaptation that is acceptable before fidelity is undermined. [ABSTRACT FROM AUTHOR]

Abstract Little is known about the exact genes that confer vulnerability or resilience to environmental stressors during early neurodevelopment. Partial genetic deletion of neuregulin 1 (Nrg1) moderates the neurobehavioural effects of stressors applied in adolescence and adulthood, however, no study has yet examined its impact on prenatal stress. Here we examined whether Nrg1 deficiency in mice modulated the impact of prenatal stress on various behaviours in adulthood. Male heterozygous Nrg1 mice were mated with wild-type female mice who then underwent daily restraint stress from days 13 to 19 of gestation. Surprisingly, prenatal stress had overall beneficial effects by facilitating sensorimotor gating, increasing sociability, decreasing depressive-like behaviour, and improving spatial memory in adulthood. Such benefits were not due to any increase in maternal care, as prenatal stress decreased nurturing of the offspring. Nrg1 deficiency negated the beneficial behavioural effects of prenatal stress on all measures except sociability. However, Nrg1 deficiency interacted with prenatal stress to trigger locomotor hyperactivity. Nrg1 deficiency, prenatal stress or their combination failed to alter acute stress-induced plasma corticosterone concentrations. Collectively these results demonstrate that Nrg1 deficiency moderates the effects of prenatal stress on adult behaviour, but it does so in a complex, domain-specific fashion. Highlights • Prenatal stress (PS) had a beneficial effect on various behaviours in adult mice. • PS improved sensorimotor gating, sociability, spatial memory and stress coping. • Nrg1 deficiency reduced all the benefits of PS except for enhanced sociability. • Nrg1 deficiency and prenatal stress combined to trigger locomotor hyperactivity. [ABSTRACT FROM AUTHOR]

Objective: To identify why the National Clinical Guideline recommendation of 45 minutes of each appropriate therapy daily is not met in many English stroke units. Design: Mixed-methods case-study evaluation, including modified process mapping, non-participant observations of service organisation and therapy delivery, documentary analysis and semi-structured interviews. Setting: Eight stroke units in four English regions. Subjects: Seventy-seven patients with stroke, 53 carers and 197 stroke unit staff were observed; 49 patients, 50 carers and 131 staff participants were interviewed. Results: Over 1000 hours of non-participant observations and 433 patient-specific therapy observations were undertaken. The most significant factor influencing amount and frequency of therapy provided was the time therapists routinely spent, individually and collectively, in information exchange. Patient factors, including fatigue and tolerance influenced therapists' decisions about frequency and intensity, typically resulting in adaptation of therapy rather than no provision. Limited use of individual patient therapy timetables was evident. Therapist staffing levels were associated with differences in therapy provision but were not the main determinant of intensity and frequency. Few therapists demonstrated understanding of the evidence underpinning recommendations for increased therapy frequency and intensity. Units delivering more therapy had undertaken patient-focused reorganisation of therapists' working practices, enabling them to provide therapy consistent with guideline recommendations. Conclusion: Time spent in information exchange impacted on therapy provision in stroke units. Reorganisation of therapists' work improved alignment with guidelines. [ABSTRACT FROM AUTHOR]

The endocannabinoid system is dysregulated in schizophrenia. Mice with heterozygous deletion of neuregulin 1 ( Nrg1 HET mice) provide a well-characterised animal model of schizophrenia, and display enhanced sensitivity to stress and cannabinoids during adolescence. However, no study has yet determined whether these mice have altered brain endocannabinoid concentrations. Nrg1 application to hippocampal slices decreased 2-arachidonoylglycerol (2-AG) signalling and disrupted long-term depression, a form of synaptic plasticity critical to spatial learning. Therefore we specifically aimed to examine whether Nrg1 HET mice exhibit increased 2-AG concentrations and disruption of spatial learning. As chronic stress influences brain endocannabinoids, we also sought to examine whether Nrg1 deficiency moderates adolescent stress-induced alterations in brain endocannabinoids. Adolescent Nrg1 HET and wild-type (WT) mice were submitted to chronic restraint stress and brain endocannabinoid concentrations were analysed. A separate cohort of WT and Nrg1 HET mice was also assessed for spatial learning performance in the Morris Water Maze. Partial genetic deletion of Nrg1 increased anandamide concentrations in the amygdala and decreased 2-AG concentrations in the hypothalamus. Further, Nrg1 HET mice exhibited increased 2-AG concentrations in the hippocampus and impaired spatial learning performance. Chronic adolescent stress increased anandamide concentrations in the amygdala, however, Nrg1 disruption did not influence this stress-induced change. These results demonstrate for the first time in vivo interplay between Nrg1 and endocannabinoids in the brain. Our results demonstrate that aberrant Nrg1 and endocannabinoid signalling may cooperate in the hippocampus to impair cognition, and that Nrg1 deficiency alters endocannabinoid signalling in brain stress circuitry. [ABSTRACT FROM AUTHOR]

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro , suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells. [ABSTRACT FROM AUTHOR]

Purpose: Nurses represent the largest professional group working with stroke-survivors, but there is limited evidence regarding nurses’ involvement in post-stroke rehabilitation. The purpose of this study was to identify and explore the perspectives of nurses and other multidisciplinary stroke team members on nurses’ practice in stroke rehabilitation. Method: Q-methodological study with 63 multidisciplinary stroke unit team members and semi-structured interviews with 27 stroke unit team members.Results: Irrespective of their professional backgrounds, participants shared the view that nurses can make an active contribution to stroke rehabilitation and integrate rehabilitation principles in routine practice. Training in stroke rehabilitation skills was viewed as fundamental to effective stroke care, but nurses do not routinely receive such training. The view that integrating rehabilitation techniques can only occur when nursing staffing levels were high was rejected. There was also little support for the view that nurses are uniquely placed to co-ordinate care, or that nurses have an independent rehabilitation role.Conclusions: The contribution that nurses with stroke rehabilitation skills can make to effective stroke care was understood. However, realising the potential of nurses as full partners in stroke rehabilitation is unlikely to occur without introduction of structured competency-based multidisciplinary training in rehabilitation skills.Implications for RehabilitationMultidisciplinary rehabilitation in stroke units is a cornerstone of effective stroke care.Views of stroke unit team members on nurses’ involvement in rehabilitation have not been reported previously.Nurses can routinely incorporate rehabilitation principles in their care.Specialist competency-based stroke rehabilitation training needs to be provided for nurses as well as for allied health professionals. [ABSTRACT FROM AUTHOR]

Recent concrete proposals suggest it is possible to engineer a two-dimensional bulk phase supporting non-Abelian Fibonacci anyons out of Abelian fractional quantum Hall systems. The low-energy degrees of freedom of such setups can be modeled as ℤ3 parafermions "hopping" on a two-dimensional lattice. We use the density matrix renormalization group to study a model of this type interpolating between the decoupled-chain, triangular-lattice, and square-lattice limits. The results show clear evidence of the Fibonacci phase over a wide region of the phase diagram, most notably including the isotropic triangular-lattice point. We also study the broader phase diagram of this model and show that elsewhere it supports an Abelian state with semionic excitations. [ABSTRACT FROM AUTHOR]

Native mass spectrometry is a rapidly emerging field for characterising the structure of proteins and protein assemblies. The technique relies on electrospray ionisation (ESI) to efficiently ionise the protein analyte and transmit it into the gas phase with retention of protein structure, non-covalent protein-ligand and protein–protein interactions. In native ESI, both the ionisation efficiency and the resulting mass spectral signal is adversely effected by the presence of non-volatile inorganic salts, such as sodium chloride, which form extensive adducts with the protein ions. Consequently, there is great interest in finding experimental strategies that mitigate these phenomena. Here we report our findings that the addition of 10 mM l-serine to the ESI spray solution reduces the adverse effects of sodium adduction to proteins. In the analysis of bovine serum albumin (BSA; 66 kDa), 10 mM serine increased signal to noise ratio (S/N) ∼4 fold. This increase in sensitivity was accompanied by peak narrowing (∼10 fold), which allowed more precise assignment of molecular mass. Similar effects were observed when analysing protein complexes – serine palmitoyl transferase (SPT, a 92 kDa homodimer), enolase (a 93 kDa homodimer); and alcohol dehydrogenase (ADH, a 148 kDa tetramer). Reduction in sodium ion adduction occurs with no loss of the non-covalent protein–protein interactions, and with little effect on the overall observed charge state-distribution. As a consequence of increasing signal intensity, the addition of serine to the ESI spray solution greatly improved the quality of the data obtained from native top-down electron-capture dissociation (ECD) experiments. In ECD analysis of native BSA, we observed an increasing in the S/N of all ECD fragments upon addition of 10 mM l-serine. The number of ECD fragments we observed with S/N ＞ 1.5 increased from 15 to 44 and the number of assigned c and z ions increased from 5 to 16. Finally we show that this phenomenon is not specific to l-serine, and occurs with several amino acids such as l-alanine. Our findings suggest that desalting may occur via binding of sodium ion to the amino acid in solution. This simple and inexpensive strategy has broad utility for improving the mass spectra obtained in a range of MS-based structural proteomic studies. [ABSTRACT FROM AUTHOR]

The fractional quantum Hall effect and superconductivity, remarkable phenomena in their own right, can harbour even more exotic physics at their interface. In particular, coupling quantum Hall edges with a superconductor can create emergent excitations known as non-Abelian anyons that trap widely coveted Majorana fermion zero-modes and generalizations thereof. We uncover non-local transport signatures of these zero-modes that not only provide striking experimental signatures of the anyons, but moreover allow one to construct novel circuit elements, including superconducting current and voltage mirrors, fractional charge transistors and flux-based capacitors. Underlying this unusual transport is a phenomenon that we term 'perfect Andreev conversion'-whereby quasiparticles propagating chirally at the edge reverse their electric charge as a result of hybridization with the zero-modes. Our findings suggest numerous experimental directions in the study of quantum-Hall-superconductor systems hybrids and highlight a fundamentally new application of non-Abelian anyons. [ABSTRACT FROM AUTHOR]

Aims and objectives To identify and synthesise the available research evidence in order to generate an explanatory framework for nursing practice in stroke rehabilitation. Background Although nurses are the largest professional group working with stroke survivors, there is limited understanding of nursing practice in stroke units. In particular, there is currently very little evidence in respect of nurses' involvement in poststroke rehabilitation. Design Meta-ethnography. Method A systematic review was undertaken. The review question was: 'What is the nature of nursing practice in the care and rehabilitation of inpatient stroke survivors?' Searches of 12 electronic databases identified 14,655 publications, and after screening, 778 remained; 137 papers were obtained and 54 retained for mapping. Sixteen qualitative studies were included in the meta-ethnography. Results Nurses' involvement in poststroke rehabilitation was limited. Contextual factors impacted on nurses' perceptions and practice. Nurses' integration of rehabilitation skills was perceived to be contingent on adequate nurse staffing levels and management of demands on nurses' time. Team working practices and use of the built environment indicated separation of nursing and therapy work. Physical care and monitoring were prioritised. Stroke-specific education and training was evident, but not consistent in content or approach. Stroke survivors and families needed help to understand nurses' role in rehabilitation. Conclusion The review provides compelling evidence that there is an need to re-examine the role of nurses in contributing to poststroke rehabilitation, including clarifying when this process can safely begin and specifying the techniques that can be integrated in nurses' practice. Relevance to clinical practice Integrating stroke-specific rehabilitation skills in nurses' practice could contribute substantially to improving outcomes for stroke survivors. The explanatory framework developed from the review findings identifies issues which will need to be addressed in order to maximise nurses' contribution to the rehabilitation of stroke survivors. [ABSTRACT FROM AUTHOR]

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of Lserine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition [ABSTRACT FROM AUTHOR]

Co-ordinated multidisciplinary team working has made a significant contribution to improvements in the quality of care within stroke services over the past five years. Here, Dr Clarke reviews the evidence for the efficacy of multi disciplinary team care in stroke rehabilitation within both inpatient services and community settings. [ABSTRACT FROM AUTHOR]

At some stage in the planning process, teachers develop or select tasks which prompt the type of activity that can offer students experiences that can help them learn the intended content. This article presents results from an item on a survey that invited teachers to match particular content descriptions with selected tasks. While many teachers matched the tasks with the content descriptions that we anticipated they would, there were also many teachers who chose descriptions that we consider do not match the tasks. It is suggested that teachers should discuss within their planning teams the purpose and potential of particular tasks, and that such discussions also form part of teacher professional learning. It is recommended that the notion of matching tasks to the curriculum is incorporated into teacher learning sessions. [ABSTRACT FROM AUTHOR]

We are interested in exploring the processes teachers use when planning. We sought responses from teachers on aspects of their planning from a survey of various aspects of their decisions. We found that, while there is diversity in processes teachers use for planning, a consistent theme was that teachers make deliberative decisions at all stages in the planning process. Likewise teachers make active use of assessment information, especially to address the needs of their students. There are important implications from our findings for those who support teachers in the transition to the Australian Curriculum: Mathematics. [ABSTRACT FROM AUTHOR]

In recent years, there has been greater attention in research and professional development work to discussion of the kinds of knowledge teachers need to teach effectively in the mathematics classroom. It seems reasonable that in planning at the school level, teaching team level and then specifically for a given lesson, teachers might have a sense of the most important mathematical ideas on which they wish to focus. In this article, we discuss written statements by teachers in surveys regarding the most important ideas on which they intended to focus in their next teaching topic. There was considerable variation in teachers' capacity to articulate these important ideas. We discuss the implications of this for teacher professional learning and planning. [ABSTRACT FROM AUTHOR]

Essential to teachers' planning are decisions regarding what should be taught. Curriculum documents are the most obvious authority. But what is a 'curriculum document' for a mathematics teacher in Australia? Are there other credible sources of information that Australian teachers draw on when deciding what to teach? This article examines the responses of a national sample of Australian teachers to a survey examining their planning practices. [ABSTRACT FROM AUTHOR]

The Australian Curriculum: Mathematics encourages teachers to consider seriously the four proficiencies: Understanding, Fluency, Problem Solving and Reasoning. In responding to the reasoning proficiency, many teachers may find that the language of the classroom may well change. In this article, we discuss the meaning given to the term reasoning within the AC:M and elsewhere, share an example of a classroom task with the potential to develop mathematical reasoning, consider the language involved in describing reasoning, and present some survey responses on teachers' current understanding and use of the relevant terms. [ABSTRACT FROM AUTHOR]

Summary The bacterium, Photorhabdus luminescens, is an insect pathogen that also maintains a mutualistic interaction with nematodes from the family Heterorhabditis. Photorhabdus luminescens is carried in the gut of the infective juvenile (IJ), a nematode stage that infects soft-cuticled insect larvae in the soil. Photorhabdus luminescens is released into the insect blood to convert the insect into a biomass that is able to support nematode growth and development. Nematode reproduction continues for 2-3 generations before the nematodes enter an alternative developmental pathway leading to a new generation of IJs that are efficiently colonized by P. luminescens in a process called transmission. Transmission begins with the adherence of P. luminescens to the rectal gland cells (RGC) located in the gut of the hermaphrodite. The bacteria enter, and replicate within, the RGC resulting in vacuolization, and ultimately lysis, of the RGC. In this way P. luminescens are released throughout the body cavity of the hermaphrodite to encounter, and colonize, the developing IJs. In this study we show that the LysR-type regulator, HdfR, is required for normal transmission in P. luminescens TTO1. Transcriptome analysis revealed that HdfR regulates the expression of 124 genes, including genes involved in arginine metabolism, hydroxyphenylacetate catabolism and pigment production. Using fluorescence microscopy we show that the ΔhdfR mutant is able to attach to, and grow within, the hermaphrodite as well as wild-type bacteria but subsequent steps in transmission are delayed. Therefore, HdfR plays an important role in coordinating the interaction between P. luminescens and its nematode partner during transmission. [ABSTRACT FROM AUTHOR]

This study attends to the possibilities of analyzing students' writings at a lexicogrammatical level in terms of their use of linguistic resources, and the insights afforded by this approach into the challenges students faced when they employed the language of school science related to the phenomenon of expansion. For the purpose of this study, the data consist of Grade 7 students' written assignments generated from a sequence of lessons on the topic of 'States of Matter.' We analyzed the language by employing the Systemic Functional Linguistics framework. The analysis reveals similarities and differences in the use of linguistic resources among the students, from which we identified several patterns of language use that appeared to be significant for realizing scientific meanings. These patterns of language use suggest that students face challenges not just in learning to use language as both a cognitive and a semiotic tool but also in interpreting the requirements of task when employing the language of school science. The findings demonstrate the productivity of this approach in expanding our understanding of the conceptual and representational challenges that students face when learning the language of school science. © 2011 Wiley Periodicals, Inc. Sci Ed 95:852-876, 2011 [ABSTRACT FROM AUTHOR]

Heterostructures of spin-orbit coupled materials with s-wave superconductors are thought to be capable of supporting zero-energy Majorana bound states. Such excitations are known to obey non-Abelian statistics in two dimensions, and are thus relevant to topological quantum computation (TQC). In a one-dimensional system, Majorana states are localized to phase boundaries. In order to bypass the constraints of one dimension, a wire network may be created, allowing the exchange of Majoranas by way of junctions in the network. Alicea et al. have proposed such a network as a platform for TQC, showing that the Majorana bound states obey non-Abelian exchange statistics even in quasi-one-dimensional systems. Here we show that the particular realization of non-Abelian statistics produced in a Majorana wire network is highly dependent on the local properties of individual wire junctions. For a simply connected network, the possible realizations can be characterized by the chirality of individual junctions. There is in general no requirement for junction chiralities to remain consistent across a wire network. We show how the chiralities of different junctions may be compared experimentally and discuss the implications for TQC in Majorana wire networks. [ABSTRACT FROM AUTHOR]

Photorhabdus luminescens is a species of Gram-negative bacteria that is pathogenic to insects while also maintaining a mutualistic association with nematodes from the family Heterorhabditis. P. luminescens elaborates an extensive secondary metabolism during the post-exponential phase of growth that includes the production of an antibiotic called 3-5-dihydroxy-4-isopropylstilbene (ST), an anthraquinone pigment (AQ) and bioluminescence. In this study we identified a mutant that was unable to produce ST, AQ and light. This mutation was found to be in the mdh gene, encoding malate dehydrogenase, a key enzyme in the tricarboxylic acid (TCA) cycle. Interestingly the mdh mutant was unaffected in virulence but was unable to support nematode growth and development in vivo or in vitro. This clearly establishes that secondary metabolism in P. luminescens is required for the mutualistic interaction with the nematode. Furthermore, the construction of mutations in key genes in other central metabolic pathways confirmed the critical role for the TCA cycle in both secondary metabolism and mutualism, but not in virulence. Therefore, we conclude that the TCA cycle is required for the transition of P. luminescens from pathogen to mutualist implicating the involvement of a metabolic switch in the regulation of lifestyle decisions in this bacterium. [ABSTRACT FROM AUTHOR]

Collaborative interdisciplinary working is central to contemporary health policy. The specialized and co-ordinated multidisciplinary care provided in stroke units is considered to contribute to improved patient outcomes in such units. However, how stroke unit teams co-ordinate their work is not clearly understood. This paper reports on a grounded theory study which explains how health professionals in two stroke units in northern England achieved teamwork. Data were generated through 220 hours of participant observation and 34 semi-structured interviews. Interviews were undertaken during and following participant observations. A basic social process common to teamworking in both units was identified; this was termed “opportunistic dialogue”. The division of labour in respect of rehabilitation activities was negotiated through this interactional process. Co-location of most team members led to repeated engagement in sharing patient information and in exploring different perspectives. Opportunistic dialoguing contributed to mutual learning and explained the shift in thinking and team culture as team members moved from concern with discrete disciplinary actions to dialogue and negotiations focused on meeting patients' needs. The findings indicate that routinely incorporating periods of joint working in which team members articulate the reasoning for their decisions and interventions, contributes to achieving interdisciplinary teamworking in rehabilitation settings. [ABSTRACT FROM AUTHOR]

We have developed an autotuated quench-flow microreactor which interfaces directly to an electiuspray ionization mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FTICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme using top-down fragmentation. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assighed with high confidence. Electron capture dissociation (ECD) fragmentation al kinetic lowed us to locate the intennediate to a five amino acid section of the protein-which includes the known catalytic residue, Ser195. This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms, is a potentially powerful tool for studies of enzyme catalysis. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such per- oxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which tacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity. [ABSTRACT FROM AUTHOR]

Abstract: We report high resolution spectra for the medium molecular weight proteins myoglobin and cytochrome-c obtained using a custom desorption electrospray ionisation (DESI) source coupled to a Bruker Daltonics 12T Apex Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The DESI source was designed for accurate alignment and reproduction of critical geometric variables. A two axis motorised stage was included to enable automated rastering of the sample under the DESI plume. Spectra for the intact proteins have been obtained under single-acquisition conditions and a top-down analysis of cytochrome-c was performed using both collision induced dissociation (CID) and electron capture dissociation (ECD) of the isolated [M+15H]15+ charge state. The sequence coverage is comparable to that obtained using electrospray ionisation, demonstrating the utility of top-down protein analysis by DESI FTICR-MS. [Copyright &y& Elsevier]

Background: Qualitative observational methods are an effective means of detailing social processes and phenomena that constitute the day-to-day reality of practice in rehabilitation units. Of vital importance in this approach is the need to record and analyse fieldnotes in sufficient detail, to ensure the researchers' explanations of particular processes or events are an accurate and credible representation of those phenomena. Content: In this second article, the process of participant observation is discussed, and the approaches to recording fieldnotes and working with qualitative observational data are described with illustrative examples. These examples also serve to highlight three principles that researchers should pay attention to when developing fieldnote records. Conclusions: Participant observation allows the researcher to gain access to and develop understanding of what the social and occupational world feels and looks like to those who work in rehabilitation. For observational data to be comprehensive and address the research questions posed, the context and organization of social settings as well as phenomena of particular interest should be captured as fully as possible. A considered and rigorous approach to recording and analysing fieldnotes will ensure this is achieved. [ABSTRACT FROM AUTHOR]

Background: Research in rehabilitation practice commonly aims to evaluate the effectiveness of complex interventions, and qualitative research-often as part of a mixed methods approach-is being increasingly employed to address important research questions in rehabilitation. Such questions often focus on the activities, experiences and perceptions of patients, carers and practitioners, as well as on the outcomes of rehabilitation. Qualitative observational methods can provide detailed accounts of social processes, which are often hidden or taken for granted, but which constitute the day-to-day reality of rehabilitation practice. Content: This two-part article examines the characteristics of systematic qualitative observational methods in the context of rehabilitation research. The first article will introduce this methodology, discuss its merits, philosophical and methodological influences, and then illustrate how qualitative observational research should be planned and conducted. The next article will discuss participant observation, and explore the approaches to recording fieldnotes and working with qualitative observational data. Conclusions: Increasingly researchers and practitioners are recognizing that reliance on traditional quantitative approaches alone cannot fully account for the all of the individual, social and organizational factors which may have a bearing on the outcomes of rehabilitation interventions. Qualitative observation is an approach available to researchers to understand the impact of these complex factors. [ABSTRACT FROM AUTHOR]

Bacterioferritin comigratory protein (BCP) is a bacterial thioredoxin-dependent thiol peroxidase that reduces a variety of peroxide substrates. Using high-resolution Fourier transform ion cyclotron resonance mass spectrometry coupled with top-down fragmentation techniques, we have analyzed the mechanistic details of hydrogen peroxide reduction by E. co/i BCP. We show here that catalysis occurs via an atypical two-cysteine peroxiredoxin pathway. A transient sulfenic acid is initially formed on Cys-45, before resolution by the formation of an intramolecular disulfide bond between Cys-45 and Cys-50. This oxidized BCP intermediate is shown to be a substrate for reduction by thioredoxin, completing the catalytic cycle. Although we invoke Cys-50 in the catalytic cycle of Escherichia co/i bacterioferritin comigratory protein (BCP), a previous study had shown that this residue was not absolutely required for peroxiredoxin activity. In order to explain these apparently conflicting phenomena, we analyzed the reaction of a C5OS BCP mutant with peroxide. We show that this mutant BCP enzyme adopts a different and novel mechanistic pathway. The C5OS BCP mutant reacts with peroxide to form a sulfenic acid on Cys-45, in the same manner as wild-type BCP. However, the nascent intermediate is then resolved by reaction with Cys-45 from a second BCP molecule, resulting in a dimeric intermediate containing an intermolecular disulfide bond. We further show that this novel resolving complex is a substrate for reduction by thioredoxin. The importance of our results in furthering the understanding of catalysis, within BCP family is discussed. [ABSTRACT FROM AUTHOR]

Photorhabdus are entomopathogenic members of the family Enterobacteriaceae. In addition to killing insects Photorhabdus also have a mutualistic association with nematodes from the family Heterorhabditidiae. Therefore, the bacteria have a complex life cycle that involves temporally separated pathogenic and mutualistic associations with two different invertebrate hosts. This tripartite Photorhabdus–insect–nematode association provides researchers with a unique opportunity to characterize the prokaryotic contribution to two different symbioses, i.e. pathogenicity and mutualism while also studying the role of the host in determining the outcome of association with the bacteria. In this review I will outline the life cycle of Photorhabdus and describe recent important advances in our understanding of the symbiology of Photorhabdus. Finally, the contribution made by this model to our understanding of the nature of symbiotic associations will be discussed. [ABSTRACT FROM AUTHOR]

β-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but eβential for chemoattractant ability. However, here we show that HBD3 (human β-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (CysV) of the β-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (CysI) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding CysV. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of β-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design. [ABSTRACT FROM AUTHOR]

Background: Photorhabdus are motile members of the family Enterobactericeae that are pathogenic to insect larvae whilst also maintaining a mutualistic interaction with entomophagous nematodes of the family Heterorhabditiae. The interactions between Photorhabdus and its hosts are thought to be an obligate part of the bacteria's life-cycle in the environment. Motility often plays a key role in mediating bacteria-host interactions and, in this study, we were interested in characterising the role of motility in the Photorhabdus-nematode-insect tripartite association. Results: We constructed deletion mutants of flgG (blocking flagella production) and motAB (blocking flagella rotation) in P. luminescens TT01. Using these mutants we show that both the ΔflgG and ΔmotAB mutants are equally as good as the wild-type (WT) bacteria in killing insects and supporting nematode growth and development suggesting that flagella production and motility are not required for pathogenicity or mutualism. However we show that the production of flagella is associated with a significant metabolic cost during growth on agar plates suggesting that, although not required for pathogenicity or mutualism, there must be a strong selective pressure to retain flagella production (and motility) during the interactions between Photorhabdus and its different hosts. To this end we show that both the ΔflgG and ΔmotAB mutants are out-competed by WT Photorhabdus during prolonged incubation in the insect revealing that motile bacteria do have a fitness advantage during colonisation of the insect larva. Conclusion: This is the first report of a role for motility in Photorhabdus and we show that, although not required for either pathogenicity or mutualism, motility does contribute to the competitive fitness of Photorhabdus during infection of the insect (and, to a lesser extent, the nematode). This adaptive function is similar to the role ascribed to motility in mammalian pathogens such as uropathogenic Escherichia coli (UPEC). Therefore, in addition to describing a role for motility in Photorhabdus, this study reinforces the relevance and utility of this bacterium as a model for studying bacteria-host interactions. [ABSTRACT FROM AUTHOR]

Photorhabdus and Xenorhabdus bacteria colonize the intestines of the infective soil-dwelling stage of entomophagous nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect susceptible insect larvae and release the bacteria into the insect blood. The bacteria kill the insect larvae and convert the cadaver into a food source suitable for nematode growth and development. After several rounds of reproduction the nematodes are recolonized by the bacteria before emerging from the insect cadaver into the soil to search for a new host. Photorhabdus and Xenorhabdus bacteria therefore engage in both pathogenic and mutualistic interactions with different invertebrate hosts as obligate components of their life cycle. In this review we aim to describe current knowledge of the molecular mechanisms utilized by Photorhabdus and Xenorhabdus to control their host-dependent interactions. Recent work has established that there is a trade-off between pathogenicity and mutualism in both these species of bacteria suggesting that the transition between these interactions must be under regulatory control. Despite the superficial similarity between the life cycles of these bacteria, it is now apparent that the molecular components of the regulatory networks controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are very different. [ABSTRACT FROM AUTHOR]

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a β-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Deft1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mM NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have β-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding (SSB) proteins stabilize single- stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full- length protein (147 amino acids; Mr 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)68]. All crystals diffract to around 2.9 Å resolution and those without bound DNA (native) belong to space group P21, with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b 73.40, c = 109.76 Å, β = 95.11°. Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 Å and could belong to three closely related space groups (I222, I212121 or I41) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way. [ABSTRACT FROM AUTHOR]

Bacteria are often found associated with surfaces as sessile bacterial communities called biofilms, and the formation of a biofilm can be split up into different stages each requiring the expression of specific genes. The production of extracellular polysaccharides (EPS) is important for the maturation of biofilms and is controlled by the Rcs two-component pathway in Escherichia coli (and other Gram-negative bacteria). In this study, we show, for the first time, that the RcsC sensor kinase is required for normal biofilm development in E. coli. Moreover, using a combination of DNA macroarray technology and transcriptional fusion analysis, we show that the expression of > 150 genes is controlled by RcsC in E. coli. In silico analyses of the RcsC regulon predicts that 50% of the genes encode proteins that are either localized to the envelope of E. coli or have activities that affect the structure/properties of the bacterial surface, e.g. the production of colanic acid. Moreover, we also show that RcsC is activated during growth on a solid surface. Therefore, we suggest that the RcsC sensor kinase may play an important role in the remodelling of the bacterial surface during growth on a solid surface and biofilm formation. [ABSTRACT FROM AUTHOR]

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein–protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCPΔ67) from the extreme hyperthermophile Aquifex aeolicus . In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCPΔ67 at temperatures of up to 70 °C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCPΔ67 complex. Furthermore, this complex was also formed between BPL and a BCCPΔ67 mutant lacking the lysine residue (BCCPΔ67 K117L) however, complex formation was considerably reduced using holo-BCCPΔ67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCPΔ67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition. [ABSTRACT FROM AUTHOR]

Summary Photorhabdus is a genus of entomopathogenic Gram-negative bacteria that belong to the family Enterobactericeae. Remarkably, at the same time as being pathogenic to insect larvae, Photorhabdus also have a mutualistic relationship with entomophagous nematodes of the family Heterorhabditiae. Photorhabdus can be isolated in two phenotypically distinct forms, termed the primary and secondary variant. Both variants grow equally well and are equally virulent when injected into insect larvae. However, only the primary variant can colonize the intestinal tract of the IJ stage of the nematode and support nematode growth and development. The primary variant expresses several phenotypes that are absent from the secondary variant, including the production of extracellular enzymes, pigments, antibiotics and light. In this study, we use Photorhabdus temperata strain K122 to show that these primary-specific products are symbiosis factors, i.e. factors that are required for nematode growth and development. We also show that, in P. temperata K122, the production of these symbiosis factors is repressed in the secondary variant by the protein encoded by a gene with homology to hexA from Erwinia . Moreover, the derepression of the symbiosis factors in the secondary variant results in a significant attenuation of virulence to larvae of the greater wax moth, Galleria mellonella . This suggests that, during a normal infection, pathogenicity and symbiosis must be temporally separated and that HexA is involved in the regulation of this pathogen–symbiont transition. [ABSTRACT FROM AUTHOR]

Native mass spectrometric analysis of TPR2A and GrpE with unpurified peptides derived from limited proteolysis of their respective PPI partners (HSP90 C-terminus and DnaK) facilitated efficient, qualitative identification of interfacial epitopes involved in transient PPI formation. Application of this approach can assist in elucidating interfaces of currently uncharacterised transient PPIs. [ABSTRACT FROM AUTHOR]

Carboxylic ester hydrolases with the capacity to degrade polyesters are currently highly sought after for their potential use in the biological degradation of PET and other chemically synthesized polymers. Here, we describe MarCE, a carboxylesterase family protein identified via genome mining of a Maribacter sp. isolate from the marine sponge Stelligera stuposa. Based on phylogenetic analysis, MarCE and its closest relatives belong to marine‐associated genera from the Cytophaga–Flavobacterium–Bacteroides taxonomic group and appear evolutionarily distinct to any homologous carboxylesterases that have been studied to date in terms of structure or function. Molecular docking revealed putative binding of BHET, a short‐chain PET derivative, onto the predicted MarCE three‐dimensional structure. The synthetic ester‐degrading activity of MarCE was subsequently confirmed by MarCE‐mediated hydrolysis of 2 mM BHET substrate, indicated by the release of its breakdown products MHET and TPA, which were measured, respectively, as 1.28 and 0.12 mM following 2‐h incubation at 30°C. The findings of this study provide further insight into marine carboxylic ester hydrolases, which have the potential to display unique functional plasticity resulting from their adaptation to complex and fluctuating marine environmentsw. [ABSTRACT FROM AUTHOR]

Examines student journal writing in mathematics. Study of four years use of journal writing in mathematics involving 500 students in grades seven through eleven; demonstrates link between language and mathematics; suggests a relationship between students' mathematical writings and their perception of mathematics and mathematical activity.

1. Vesicles which demonstrated ATP-dependent proton influx were produced from cell membranes of Clostridium pasteurianum by a cholate-dialysis procedure which was also employed to introduce bacteriorhodopsin into such membrane vesicles and into artificial proteoliposomes. 2. ATP synthetase activity was assayed using illuminated bacteriorhodopsin-containing crude membrane vesicles plus a glucose and hexokinase ‘ATP trap’. The membrane-bound ATPase of vegetatively grown cells of Cl. pasteurianum displayed measurable ATP synthetase activity in this assay. 3. ATPase-proteoliposomes constructed of purified ATPase (BF0F1) of Cl. pasteurianum with bacteriorhodopsin and a mixture of phospholipids accomplished light-dependent synthesis of ATP from ADP plus Pi. The reaction was inhibited by N,N′-dicyclohexylcarbodiimide and by proton conductors such as tetrachlorosalicylanilide. The specific ATP synthetase activity of the purified Cl. pasteurianum ATPase was significantly less than that of similarly purified ATPases (BF0F1) from Escherichia coli, Streptococcus faecalis and Streptococcus pleornorphus. The specific ATP synthetase activity of the ATPase of Clostridium formicoaceticum was greater when the enzyme complex was derived from fumarate-grown cells then when it was purified from organisms grown on fructose. 4. The apparent Km value (for Mg2+-ADP-Pi) displayed by the ATPase of Cl. pasteurianum when acting as an ATP synthetase was much higher than the apparent Km value (for ATP) in ATP phosphohydrolysis. A similar disposition to serve as an ATP phosphohydrolase was displayed by the ATPase of fructose-grown Cl. formicoaceticum, but the ATPase from fumarate-grown cells of this organism was substantially more effective in ATP synthesis. 5. The ATP synthetase activity of Cl. pasteurianum ATPase (BF0F1) was as susceptible as was its ATP phosphohydrolase activity to inhibition by dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, quercetin and citreoviridin and was similarly insensitive to inhibition by triethyl tin and tributyl tin. [ABSTRACT FROM AUTHOR]

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N′-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N′-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (± 10%) as follows: subunit a, 65500; subunit c, 57500; subunit da, 43000; subunit fa, 15000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N′-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-l-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone), by valinomycin in the presence of a high external concentration of K+, or by N,N′-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component. [ABSTRACT FROM AUTHOR]

1. The apparent transmembrane bulk-phase electrical potential (Δψ)of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolysing cells the highest value of Δψ, calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2-3 mM K+ ions and, even so, was only 100-110 mV. 2. Efrapeptin, a BF1-directed inhibitor of the membrane H+-ATPase of Cl. pasteurianum, abolished the membrane potential (Δψ) and caused complete efflux of actively-transported K+ ions. Thus protonmotive hydrolysis of ATP generated by substrate level phosphorylation is the sole means of membrane energisation in this anaerobe. 3. At low (sublethal) concentrations, butyricin 7423 stimulated K+ efflux from Cl. pasteurianum without measurably affecting its membrane potential. At lethal and supralethal concentrations of this bacteriocin, both Δψ and active K+ uptake were abolished. 4. Whilst the addition of valinomycin to cells of Cl. pasteurianum suspended in media of low K+ concentration generated a diffusion potential to which BuPh3P+ would respond, addition of butyricin 7423 in place of valinomycin caused no such effect. Also, unlike valinomycin, butyricin 7423 did not increase the rate of K+ efflux from nonglycolysing cells of Cl. pasteurianum. Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolysing cells of Cl. pasteurianum. 5. A mutant strain of Cl. pasteurianum (viz. strain DC3) which possessed a H+-ATPase with diminished sensitivity both to N,N'-dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in Δψ and in K+ accumulation ratio in response to concentrations of butyricin 7423 that were bactericidal to the wild-type, parent organism. Even so, the bactericidal action of butyricin 7423 on Cl. pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism. 6. Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na+ ions from Cl. pasteurianum nor exhibited any protonophorous activity. However, at artificially high concentration, butyricin 7423 catalysed the passage of Na+ ions as well as of K+ ions through multilayer lipid membranes. 7. As a non-protonophorous uncoupler, butyricin 7423 appears to act in a similar manner to that of the membrane-active colicins. Yet no evidence was obtained that butyricin 7423 at its minimum lethal concentration might form a gated ion channel in the cytoplasmic membrane of the target cell, or act as a classic ionophore. [ABSTRACT FROM AUTHOR]

The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) is required for de novo sphingolipid biosynthesis. A previous study revealed a novel and unexpected interaction between the hydroxyl group of the l-serine substrate and the 5′-phosphate group of PLP. By using pyridoxal (PL), the dephosphorylated analogue of vitamin B6, we show here that this interaction is important for substrate specificity and optimal catalytic efficiency. [ABSTRACT FROM AUTHOR]

Abstract: Oxidation of cysteine is now known to serve as a fundamental mechanism to control protein function or activity. Many redox-regulated proteins do not oxidize to homogeneity, resulting in a mixture of reduced and oxidized species which cannot be separated chromatographically. Here we describe a protocol for the separation of reduced and oxidized forms of the tumor suppressor protein p53. This purification method relies on the reversible labeling of thiol groups with biotin and exploitation of the ultrastrong biotin–avidin interaction. This purification procedure can be applied to other cysteine-containing proteins where enrichment of the oxidized form is required. [Copyright &y& Elsevier]

Lignin is a complex heteroaromatic polymer which is one of the most abundant and diverse biopolymers on the planet. It comprises approximately one third of all woody plant matter, making it an attractive candidate as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. However, the inherent complexity of lignin makes it difficult to analyse and characterise using common analytical techniques, proving a hindrance to the utilisation of lignin as a green chemical feedstock. Herein we outline the tracking of lignin degradation by an alkaliphilic laccase in a semi-quantitative manner using a combined chemical analysis approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to characterise shifts in chemical diversity and relative abundance of ions, and NMR to highlight changes in the structure of lignin. Specifically, an alkaliphilic laccase was used to degrade an industrially relevant lignin, with compounds such as syringaresinol being almost wholly removed (95%) after 24 hours of treatment. Structural analyses reinforced these findings, indicating a >50% loss of NMR signal relating to β-β linkages, of which syringaresinol is representative. Ultimately, this work underlines a combined analytical approach that can be used to gain a broader semi-quantitative understanding of the enzymatic activity of laccases within a complex, non-model mixture. [ABSTRACT FROM AUTHOR]

The luminescence from europium doped lanthanum trifluoride (Eu3+:LaF3) nano-crystals can be greatly enhanced by capping with β-diketonate organic ligands. Here, we report on photo-stability measurements for the case of nano-crystals capped with thenoyltrifluroacetone (TTA) and compared with those capped with an inactive ligand, oleic acid. With exposure to UV pump light, we observed significant decrease in fluorescence and change in emission spectrum of the TTA-capped nano-particles whilst the fluorescence lifetime remained approximately constant. After a dose of order 70 kJ cm-2, the luminescence level was similar to that of oleic acid capped nano-crystals. We discuss possible mechanisms. [ABSTRACT FROM AUTHOR]

Spin-orbit coupled semiconducting nanowires with proximity-induced superconductivity are expected to host Majorana zero modes at their endpoints when a sufficiently strong magnetic field is applied. The resulting phase would be a one-dimensional topological superconductor. However, while a variety of experiments have been performed observing a zero-bias conductance peak (suggestive of Majorana zero modes), the topological nature of these physical systems is still a subject of debate. Here we suggest a quantitative test of the degree to which a system displaying a zero bias peak may be considered topological. The experiment is similar to previous measurements of conductance but is performed with the aid of a quantum dot at the wire's end. We arrive at the surprising result that the nonlocal behavior of a topological system may be tested through a local measurement. [ABSTRACT FROM AUTHOR]