Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis

Stakeholder involvement in systematic reviews: a protocol for a systematic review of methods, outcomes and effects

Background There is an expectation for stakeholders (including patients, the public, health professionals, and others) to be involved in research. Researchers are increasingly recognising that it is good practice to involve stakeholders in systematic reviews. There is currently a lack of evidence about (A) how to do this and (B) the effects, or impact, of such involvement. We aim to create a map of the evidence relating to stakeholder involvement in systematic reviews, and use this evidence to address the two points above. Methods We will complete a mixed-method synthesis of the evidence, first completing a scoping review to create a broad map of evidence relating to stakeholder involvement in systematic reviews, and secondly completing two contingent syntheses. We will use a stepwise approach to searching; the initial step will include comprehensive searches of electronic databases, including CENTRAL, AMED, Embase, Medline, Cinahl and other databases, supplemented with pre-defined hand-searching and contacting authors. Two reviewers will undertake each review task (i.e., screening, data extraction) using standard systematic review processes. For the scoping review, we will include any paper, regardless of publication status or study design, which investigates, reports or discusses involvement in a systematic review. Included papers will be summarised within structured tables. Criteria for judging the focus and comprehensiveness of the description of methods of involvement will be applied, informing which papers are included within the two contingent syntheses. Synthesis A will detail the methods that have been used to involve stakeholders in systematic reviews. Papers from the scoping review that are judged to provide an adequate description of methods or approaches will be included. Details of the methods of involvement will be extracted from included papers using pre-defined headings, presented in tables and described narratively. Synthesis B will include studies that explore the effect of stakeholder involvement on the quality, relevance or impact of a systematic review, as identified from the scoping review. Study quality will be appraised, data extracted and synthesised within tables. Discussion This review should help researchers select, improve and evaluate methods of involving stakeholders in systematic reviews. Review findings will contribute to Cochrane training resources

Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes

The main source of cholesterol in the central nervous system (CNS) is represented by glial cells, mainly astrocytes, which also synthesise and secrete apolipoproteins, in particular apolipoprotein E (ApoE), the major apolipoprotein in the brain, thus generating cholesterol-rich high density lipoproteins (HDLs). This cholesterol trafficking, even though still poorly known, is considered to play a key role in different aspects of neuronal plasticity and in the stabilisation of synaptic transmission. Moreover, cell cholesterol depletion has recently been linked to a reduction in amyloid beta formation. Here we demonstrate that guanosine, which we previously reported to exert several neuroprotective effects, was able to increase cholesterol efflux from astrocytes and C6 rat glioma cells in the absence of exogenously added acceptors. In this effect the phosphoinositide 3 kinase/extracellular signal-regulated kinase 1/2 (PI3K/ERK1/2) pathway seems to play a pivotal role. Guanosine was also able to increase the expression of ApoE in astrocytes, whereas it did not modify the levels of ATP-binding cassette protein A1 (ABCA1), considered the main cholesterol transporter in the CNS. Given the emerging role of cholesterol balance in neuronal repair, these effects provide evidence for a role of guanosine as a potential pharmacological tool in the modulation of cholesterol homeostasis in the brain

Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.National Institutes of Health (U.S.) (DP1 OD003961

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5′.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8′.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting

Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single-Molecule Imaging on Living Cells

We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.National Institutes of Health (U.S.) (R01 GM072670)Camille & Henry Dreyfus FoundationMassachusetts Institute of Technology. Computational and Systems Biology Program. MIT-Merck Postdoctoral Fellowshi

Spontaneous and deliberate future thinking: A dual process account

© 2019 Springer Nature.This is the final published version of an article published in Psychological Research, licensed under a Creative Commons Attri-bution 4.0 International License. Available online at: https://doi.org/10.1007/s00426-019-01262-7.In this article, we address an apparent paradox in the literature on mental time travel and mind-wandering: How is it possible that future thinking is both constructive, yet often experienced as occurring spontaneously? We identify and describe two ‘routes’ whereby episodic future thoughts are brought to consciousness, with each of the ‘routes’ being associated with separable cognitive processes and functions. Voluntary future thinking relies on controlled, deliberate and slow cognitive processing. The other, termed involuntary or spontaneous future thinking, relies on automatic processes that allows ‘fully-fledged’ episodic future thoughts to freely come to mind, often triggered by internal or external cues. To unravel the paradox, we propose that the majority of spontaneous future thoughts are ‘pre-made’ (i.e., each spontaneous future thought is a re-iteration of a previously constructed future event), and therefore based on simple, well-understood, memory processes. We also propose that the pre-made hypothesis explains why spontaneous future thoughts occur rapidly, are similar to involuntary memories, and predominantly about upcoming tasks and goals. We also raise the possibility that spontaneous future thinking is the default mode of imagining the future. This dual process approach complements and extends standard theoretical approaches that emphasise constructive simulation, and outlines novel opportunities for researchers examining voluntary and spontaneous forms of future thinking.Peer reviewe

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans