Synechocystis sp. PCC 6803, a model cyanobacterium, produces H-2 by photosystem II-independent pathway under sulfur-deprived anaerobic conditions by inactivating photosystems II. To study anaerobic H-2 photoproduction, it was grown aerobically until late exponential phase, then switched to four anaerobic substrate-stress conditions of sulfur-deprived media (SDM) only (control) or supplemented with a carbon source in autotrophic CO2 or fermentation (either glucose or acetate) conditions. Changes in H-2 photoproduction, cell growth (OD), pH, and chlorophyll content (Chl a) were monitored. Key metabolic changes and altered protein expressions were identified using an 8-plex iTRAQ-based proteomics method. Acetate as a carbon source significantly increased H-2 yield to 605.8 mu L mg Chl a(-1), but stabilized pH, and inhibited cell growth. Control conditions yielded 279 mu L mg Chl a(-1), while CO2 and glucose conditions had different yields, 72.6 and 35.7 respectively. Under (glucose) conditions, cell growth increased, but pH dropped to 4.9, reducing H-2 photoproduction. Proteomic analysis identified 1713 proteins at a false discovery rate (FDR) of <= 1 %. In acetate conditions, proteins related to glycolysis, Calvin cycle, photosystems, and pyruvate metabolism were down-regulated, suggesting inactivation of photosystems (including D1 protein (PsbA)). Notably, the absence of detectable D1 protein (a core component of PSII) in the differential expression analysis across all conditions suggests functional deactivation of PSII, leading to increased H-2 production via PSII-independent pathway and relatively stabilized Chl a content after 24 h. Glucose conditions down-regulated glycolysis and TCA cycle but upregulated photosynthesis through PSI, PSII, and RuBisCo. Despite the upregulation of H-2-inducing proteins (such as lexA, sll1626, sll0359, Ferredoxin-1, petF, and Flavodoxin) in CO2 and glucose conditions, H-2 production was lower than acetate conditions. The study offers the first comprehensive view of the molecular mechanisms of H-2 regulation, providing a list of potential gene targets for further engineering of H-2 photoproduction by Synechocystis sp. PCC 6803.

Abstract Introduction Despite significant efforts to increase deprescribing (1), marginal progress has been achieved (2). Deprescribing is a complex process involving multiple steps and activities, of which some may be routinely undertaken whilst others require interventions to support behaviour-change. Aim We aimed to develop an internationally derived specification validated by practitioners from multiple health systems that stipulates both the steps and activities required to deliver those steps of safe deprescribing. We also aimed to estimate the extent to which the required activities are currently undertaken and identify the barriers and enablers that need addressing to deliver safe deprescribing. Methods We formulated an electronic survey comprising literature reported deprescribing activities. Relevant networks in 25 countries e.g., British Geriatrics Society and Australian deprescribing network, emailed the survey link to all of their member practitioners. Respondents reported the frequency with which they thought each deprescribing activity was undertaken in practice within their peer group on a five-point Likert scale ranging from 1 (never) to 5 (always) and whether it was important. We invited extended responses regarding the barriers and enablers to deprescribing and analysed these using the Theoretical Domains Framework (TDF). Results From 263 respondents 77.9% were prescribers; 110 (41.8%) were doctors, 85 (32.3%) were pharmacists, 44 (16.7%) were nurses and 24 (9.1%) were other healthcare professionals. Eighteen activities were combined into four deprescribing steps summarised in table 1. All were considered important and clinical activities were ‘often’ or ‘always’ undertaken. Patient orientated activities were only ‘sometimes’ undertaken. The barriers and enablers requiring addressing are in the TDF domains of ‘social influence’ to support practitioners to undertake patient orientated activities and ‘environmental context and resources’ to ensure they have sufficient capacity. Conclusion An internationally derived literature and practice informed process for safe deprescribing has been established. Social desirability bias may have inflated reported frequency of activities being undertaken. We therefore chose reporting on the collective rather than own behaviour, to ameliorate these effects. Organisations should prioritise the inadequate collaboration with patients through addressing practitioners’ behavioural determinants. References 1. Scott S, Clark A, Farrow C, May H, Patel M, Twigg MJ, et al. Deprescribing admission medication at a UK teaching hospital; a report on quantity and nature of activity. International journal of clinical pharmacy. 2018;40(5):991–6. 2. O’Mahony D, Gudmundsson A, Soiza RL, Petrovic M, Cruz-Jentoft AJ, Cherubini A, et al. Prevention of adverse drug reactions in hospitalized older patients with multi-morbidity and polypharmacy: the SENATOR* randomized controlled clinical trial. Age and Ageing. 2020;49(4):605–14.

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration ( rd1 ). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.

Metal homeostasis is fundamental for optimal performance of cell metabolic pathways. Over the course of evolution, several systems emerged to warrant an intracellular metal equilibrium. When exposed to growth-challenging copper concentrations, Gram-negative bacteria quickly activate copper-detoxification mechanisms, dependent on transmembrane-protein complexes and metallochaperones that mediate metal efflux. Here, we show that vesiculation is also a common bacterial response mechanism to high copper concentrations, and that extracellular vesicles (EVs) play a role in transporting copper. We present evidence that bacteria from different ecological niches release copious amounts of EVs when exposed to copper. Along with the activation of the classical detoxification systems, we demonstrate that copper-stressed cells of the cyanobacterium Synechocystis sp. PCC6803 release EVs loaded with the copper-binding metallochaperone CopM. Under standard growth conditions, CopM-loaded EVs could also be isolated from a Synechocystis strain lacking a functional TolC-protein, which we characterize here as exhibiting a copper-sensitive phenotype. Analyses of Synechocystis tolC-mutant’s EVs isolated from cells cultivated under standard conditions indicated the presence of copper therein, in significantly higher levels as compared to those from the wild-type. Altogether, these results suggest that release of EVs in bacteria represent a novel copper-secretion mechanism, shedding light into alternative mechanisms of bacterial metal resistance.

Acetogenic bacteria are capable of fermenting CO 2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO 2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆ agrD1 ∆ agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

Microbial pretreatments have been identified as a compatible and sustainable process with anaerobic digestion compared to energy-intensive physicochemical pretreatments. In this study, barley straw and hay co-substrate was pretreated with a microaerobic barley straw-adapted microbial (BSAM) consortium prior to anaerobic digestion. The improved digestibility was investigated through 16S rRNA gene sequencing, microbial counts and C:N ratios. BSAM pretreatment resulted in 15.2 L kg(-1) TS of methane yield after 35 days, almost 40 times more than the control. The methane content in total biogas produced were 58% (v/v) and 10% (v/v) in BSAM and control, respectively. This research demonstrated that BSAM-based pretreatment significantly increased the digestibility and surface area of the lignocellulosic material and considerably enhanced biomethanation. This study generates new potential bio-research opportunities in the emerging field of lignocellulosic anaerobic digestion-biorefineries.

Hydrothermal reaction and photodeposition were used to transform TiO2 nanoparticles to TiO2 nanotubes (TNTs) loaded with Ag. The structure and morphology of TNTs and Ag/TNTs were characterized, and the antibacterial activity of all catalysts against Methicillin-resistant S. aureus was tested using agar well diffusion and total viable plate count methods in the absence and presence of UV light. The cobweb-like structure of TNTs was noticed, and the network could increase the surface area to 184-216 m(2)/g, an up to fourfold increase compared to TiO2 P25. The XRD, EDS, HRTEM and UV-DR results revealed Ag nanoparticle deposition on the TNTs surface. The HRTEM result also showed no change in the shape or morphology of TNTs. The Ag nano particle size on the TNTs surface ranged from 8.928 +/- 1.487 to 14.654 +/- 2.901 nm. The light absorption capacity of TiO2, TNTs, and Ag/TNTs displayed antibacterial activity in the presence of UV light. In contrast, only Ag/ TNTs could reduce the bacteria growth in the dark condition, which decreased the restriction of TNTs and TiO2. The maximum antibacterial activity was obtained at 1.5%Ag/TNT, at which the cell viability was 77.86% of the initial number concentration. Based on the result of this research work, it could be confirmed that Ag/TNTs can potentially be used as a disinfection material in both presence as well as absence of UV light.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

Archaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms.

There is no consensus on the best optogenetic tool for neuronal inhibition. Lamprey parapinopsin (‘Lamplight’) is a Gi/o-coupled bistable animal opsin that can be activated and deactivated by short and long wavelength light, respectively. Since native mechanisms of neuronal inhibition frequently employ Gi/o signalling, we asked here whether Lamplight could be used for optogenetic silencing. We show that short (405nm) and long (525nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, and that combining these wavelengths can be used to achieve intermediate levels of activity. We demonstrate that these properties can be applied to produce switchable and scalable neuronal hyperpolarisation, and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. We show that expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, and that 405 and 525nm stimuli can produce responses of opposite sign in output neurons of the retina. Lamplight-driven responses to both activating (405nm) and deactivating (525nm) light can occur within 500ms and be elicited by intensities at least 10x below threshold for available inhibitory optogenetic tools. We conclude that Lamplight can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and rapidly reversible.

Significant technical advancements in phosphopeptide enrichment have enabled the identification of thousands of p-peptides (mono and multiply phosphorylated) in a single experiment. However, it is still not possible to enrich all p-peptide species in a single step. A range of new techniques and materials has been developed, with the potential to provide a step-change in phosphopeptide enrichment. The first half of this review contains a tutorial for new potential phosphoproteomic researchers; discussing the key steps of a typical phosphoproteomic experiment used to investigate canonical phosphorylation sites (serine, threonine and tyrosine). The latter half then show-cases the latest developments in p-peptide enrichment including: i) Strategies to mitigate non-specific binding in immobilized metal ion affinity chromatography and metal oxide affinity chromatography protocols; ii) Techniques to separate multiply phosphorylated peptides from monophosphorylated peptides (including canonical from non-canonical phosphorylated peptides), or to simultaneously co-enrich other post-translational modifications; iii) New hybrid materials and methods directed towards enhanced selectivity and efficiency of metal-based enrichment; iv) Novel materials that hold promise for enhanced phosphotyrosine enrichment. A combination of well-understood techniques and materials is much more effective than any technique in isolation; but the field of phosphoproteomics currently requires benchmarking of novel materials against current methodologies to fully evaluate their utility in peptide based proteoform analysis.

A Correction to this paper has been published: https://doi.org/10.1007/s00253-020-11037-5

Lignin is one of three componentsthat make up wood, and it is the most recalcitrant among these compoundsdue to itshighly degradation-resistant phenolic polymerstructure. Lignin is composed of carbon,oxygen and hydrogen,which has the potential to be a feedstock for biofuels and biorefining processes. In this work, lignin was depolymerized to produce succinic and acetic acidsvia aphotocatalytic reaction. TiO2and H2O2under UV-light were used as a photocatalyst and photocatalystpromoter, respectively. The effect of TiO2and H2O2dosage, solutionpHand reaction time on %yield of dicarboxylic acid was determined. Optimizationofreaction conditionswas done withresponsesurface methodology using a Box-Behnken design. It was found that the maximum %yield of succinic acid (7.8%) was at a reaction timeof24 h, a 2.37 g/l of TiO2dosage and 25.45μl of H2O2dosage and pH 7.0. The predicted dicarboxylic acid yield using wasaccurate withR2=91.8%. This would be an alternative way to produce high-value fine chemicalsfrom lignin.

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels.

This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biological saccharification of Miscanthus giganteus (MG) for generation of fermentable saccharides and its subsequent fermentation into solvents i.e. acetone, ethanol and butanol (ABE) using Clostridium acetobutylicum ATCC 824. Saccharide hydrolysates were derived from MG by thermochemical (water, acid and alkali at 130 degrees C) and biological saccharification (Fibrobacter succinogenes S85) processes and were subjected to batch fermentation for 120 h using C. acetobutylicum ATCC 824. At the end of fermentation of thermochemically-derived hydrolysates, 742 g m(-3) of saccharides from water treatment, 9572 g m(-3) of saccharides from acid treatment and 4054 gm(-3) of saccharides from alkali treatment were fermented and yielded 0.045, 0.0069 and 0.01 g g(-1) of total solvents, respectively. Similarly, at the end of fermentation of biological hydrolysate (using F. succinogenes), 2504 gm(-3) of saccharides was fermented and yielded 0.091 g g(-1) of total solvents. The highest yield of total solvents was achieved by water (thermochemical) and biological saccharification of MG using C. acetobutylicum. Whereas, acid and alkali-treated hydrolysates showed lower yields of solvents presumably due to production of inhibitory compounds during saccharification. Compared to thermochemical saccharification, biological saccharification using F. succinogenes is a promising approach since it yielded the highest amount of solvents whilst being eco-friendly. Our future studies will focus on optimisation of biological saccharification (using F. succinogenes) and sequential co-culture fermentation (using C. acetobutylicum). The development of alternative consolidated bio-processing approach using biological saccharification will contribute towards making lignocellulosic biofuels a reality.

Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC50s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02–60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

There is an urgent need (recognized in FDA guidance, 2018) to optimize the dose of medicines given to patients for maximal drug efficacy and limited toxicity (precision dosing), which can be facilitated by quantitative systems pharmacology (QSP) models. Accurate quantification of proteins involved in drug clearance is essential to build and improve QSP models for any target population. Here we describe application of label-free proteomics in microsomes from 23 human livers to simultaneously quantify 188 enzymes and 66 transporters involved in xenobiotic disposition, including 17 cytochrome P450s (CYPs), 10 UDP-glucuronosyltransferases (UGTs), 7 ATP-binding cassette (ABC) transporters, and 11 solute carrier (SLC) transporters; six of these proteins are quantified for the first time. The methodology allowed quantification of thousands of proteins, allowing estimation of sample purity and understanding of global patterns of protein expression. There was overall good agreement with targeted quantification and enzyme activity data, where this was available. The effects of sex, age, genotype, and BMI on enzyme and transporter expression were assessed. Decreased expression of enzymes and transporters with increasing BMI was observed, but a tendency for older donors to have higher BMIs may have confounded this result. The effect of genotype on enzymes expression was, however, clear-cut, with CYP3A5*1/*3 genotype expressed 16-fold higher compared with its mostly inactive *3/*3 counterpart. Despite the complex, time-consuming data analysis required for label-free methodology, the advantages of the label-free method make it a valuable approach to populate a broad range of system parameters simultaneously for target patients within pharmacology and toxicology models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.