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Abstract 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.

Summary: Enormous efforts have been made to understand the functions of bioluminescence; however, its relevance in soil ecosystems has barely been investigated. In addition, our understanding of the biological relevance of bioluminescence is hampered by the scarcity of tools to genetically manipulate this trait. Using the symbionts of entomopathogenic nematodes, Photorhabdus bacteria, we show that bioluminescence plays important regulatory roles in multitrophic interactions in the soil. Through genetic modifications and exploiting natural variability, we provide direct evidence for the multifunctional nature of bioluminescence. It regulates abiotic and biotic stress resistance, impacts other trophic levels, including nematodes, insects, and plants, and contributes to symbiosis. Our study contributes to understanding the factors that have driven the evolution and maintenance of this trait in belowground ecosystems.

Abstract 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 The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or ‘SspA’), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.

Introduction Stroke survivors spend long periods of time engaging in sedentary behaviour (SB) even when their functional recovery is good. In the RECREATE programme, an intervention aimed at reducing SB (‘Get Set Go’) will be implemented and evaluated in a pragmatic external pilot cluster randomised controlled trial with embedded process and economic evaluations. We report the protocol for the process evaluation which will address the following objectives: (1) describe and clarify causal assumptions about the intervention, and its mechanisms of impact; (2) assess implementation fidelity; (3) explore views, perceptions and acceptability of the intervention to staff, stroke survivors and their carers; (4) establish the contextual factors that influence implementation, intervention mechanisms and outcomes.Methods and analysis This pilot trial will be conducted in 15 UK-based National Health Service stroke services. This process evaluation study, underpinned by the Medical Research Council guidance, will be undertaken in six of the randomised services (four intervention, two control). Data collection includes the following: observations of staff training sessions, non-participant observations in inpatient and community settings, semi-structured interviews with staff, patients and carers, and documentary analysis of key intervention components. Additional quantitative implementation data will be collected in all sites. Training observations and documentary analysis data will be summarised, with other observational and interview data analysed using thematic analysis. Relevant theories will be used to interpret the findings, including the theoretical domains framework, normalisation process theory and the theoretical framework of acceptability. Anticipated outputs include the following: recommendations for intervention refinements (both content and implementation); a revised implementation plan and a refined logic model.Ethics and dissemination The study was approved by Yorkshire & The Humber - Bradford Leeds Research Ethics Committee (REC reference: 19/YH/0403). Findings will be disseminated via peer review publications, and national and international conference presentations.Trial registration number ISRCTN82280581.

The chemical diversity of peptides from ribosomal origin is a growing field of research. Here, the authors report the discovery, genomic and biosynthetic investigations of kintamdin, a ribosomally synthesized and post-translationally modified peptides featuring a beta-enamino acid and a bis-thioether macrocyclic motif.

The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s−1 at 45°C, which was increased by 5-fold to 0.02 s−1 upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.

Here, the authors generated an artificial RNA molecule, or aptamer, specific for the Amyotrophic Lateral Sclerosis protein TDP-43. By interacting avidly with its target, the aptamer can be exploited to track TDP-43 phase transition in vitro and in cells.

Abstract Background Stroke survivors are highly sedentary; thus, breaking up long uninterrupted bouts of sedentary behaviour could have substantial health benefit. However, there are no intervention strategies specifically aimed at reducing sedentary behaviour tailored for stroke survivors. The purpose of this study was to use co-production approaches to develop an intervention to reduce sedentary behaviour after stroke. Methods A series of five co-production workshops with stroke survivors, their caregivers, stroke service staff, exercise professionals, and researchers were conducted in parallel in two-stroke services (England and Scotland). Workshop format was informed by the behaviour change wheel (BCW) framework for developing interventions and incorporated systematic review and empirical evidence. Taking an iterative approach, data from activities and audio recordings were analysed following each workshop and findings used to inform subsequent workshops, to inform both the activities of the next workshop and ongoing intervention development. Findings Co-production workshop participants (n = 43) included 17 staff, 14 stroke survivors, six caregivers and six researchers. The target behaviour for stroke survivors is to increase standing and moving, and the target behaviour for caregivers and staff is to support and encourage stroke survivors to increase standing and moving. The developed intervention is primarily based on co-produced solutions to barriers to achieving the target behaviour. The developed intervention includes 34 behaviour change techniques. The intervention is to be delivered through stroke services, commencing in the inpatient setting and following through discharge into the community. Participants reported that taking part in intervention development was a positive experience. Conclusions To our knowledge, this is the first study that has combined the use of co-production and the BCW to develop an intervention for use in stroke care. In-depth reporting of how a co-production approach was combined with the BCW framework, including the design of bespoke materials for workshop activities, should prove useful to other researchers and practitioners involved in intervention development in stroke.

Abstract Background Stroke survivors are more sedentary than healthy, age-matched controls, independent of functional capacity. Interventions are needed to encourage a reduction in overall sedentary time, and regular breaks in prolonged periods of sedentary behaviour. This study captured the views and experiences of stroke survivors and their caregivers related to sedentary behaviour after stroke, to inform the development of an intervention to reduce sedentary behaviour. Methods Mixed-methods qualitative study. Non-participant observations were completed in two stroke services, inclusive of inpatient and community settings in the United Kingdom. Semi-structured interviews were conducted with stroke survivors and their caregivers (if available) at six- or nine-months post-stroke. Underpinned by the capability, opportunity and motivation (COM-B) model of behaviour change, observational data (132 h) were analysed thematically and interview data (n = 31 stroke survivors, n = 12 caregivers) were analysed using the Framework approach. Results Observation participants differed in functional ability whereas stroke survivor interviewees were all ambulant. Six themes related to sedentary behaviour after stroke were generated: (1) sedentary behaviour levels and patterns after stroke; (2) the physical and social environment in the stroke service and in the home; (3) standing and movement capability after stroke; (4) emotion and motivation after stroke; (5) caregivers’ influence on, and role in influencing stroke survivors’ sedentary behaviour; and (6) intervening to reduce sedentary behaviour after stroke. Capability, opportunity and motivation were influenced by the impact of the stroke and caregivers’ inclination to support sedentary behaviour reduction. Stroke survivors reported being more sedentary than they were pre-stroke due to impaired balance and co-ordination, increased fatigue, and reduced confidence in mobilising. Caregivers inclination to support stroke survivors to reduce sedentary behaviour depended on factors including their willingness to withdraw from the caregiver role, and their perception of whether the stroke survivor would act on their encouragement. Conclusions Many stroke survivors indicate being open to reducing sedentary behaviour, with appropriate support from stroke service staff and caregivers. The findings from this study have contributed to an intervention development process using the Behaviour Change Wheel (BCW) approach to develop strategies to reduce sedentary behaviour after stroke.

Many marine bacteria produce extracellular enzymes that degrade complex molecules to facilitate their growth in environmental conditions that are often harsh and low in nutrients. Marine bacteria, including those inhabiting sea sponges, have previously been reported to be a promising source of polyesterase enzymes, which have received recent attention due to their potential ability to degrade polyethylene terephthalate (PET) plastic. During the screening of 51 marine bacterial isolates for hydrolytic activities targeting ester and polyester substrates, a Brachybacterium ginsengisoli B129SM11 isolate from the deep-sea sponge Pheronema sp. was identified as a polyesterase producer. Sequence analysis of genomic DNA from strain B129SM11, coupled with a genome “mining” strategy, allowed the identification of potential polyesterases, using a custom database of enzymes that had previously been reported to hydrolyze PET or other synthetic polyesters. This resulted in the identification of a putative PET hydrolase gene, encoding a polyesterase-type enzyme which we named BgP that shared high overall similarity with three well-characterized PET hydrolases—LCC, TfCut2, and Cut190, all of which are key enzymes currently under investigation for the biological recycling of PET. In silico protein analyses and homology protein modeling offered structural and functional insights into BgP, and a detailed comparison with Cut190 revealed highly conserved features with implications for both catalysis and substrate binding. Polyesterase activity was confirmed using an agar-based polycaprolactone (PCL) clearing assay, following heterologous expression of BgP in Escherichia coli. This is the first report of a polyesterase being identified from a deep-sea sponge bacterium such as Brachybacterium ginsengisoli and provides further insights into marine-derived polyesterases, an important family of enzymes for PET plastic hydrolysis. Microorganisms living in association with sponges are likely to have increased exposure to plastics and microplastics given the wide-scale contamination of marine ecosystems with these plastics, and thus they may represent a worthwhile source of enzymes for use in new plastic waste management systems. This study adds to the growing knowledge of microbial polyesterases and endorses further exploration of marine host-associated microorganisms as a potentially valuable source of this family of enzymes for PET plastic hydrolysis.

There has been increasing interest in bacterial lipids in recent years due, in part, to their emerging role as molecular signalling molecules. Bacteroides thetaiotaomicron is an important member of the mammalian gut microbiota that has been shown to produce sphingolipids (SP) that pass through the gut epithelial barrier to impact host SP metabolism and signal into host inflammation pathways. B. thetaiotaomicron also produces a novel family of N-acyl amines (called glycine lipids) that are potent ligands of host Toll-like receptor 2 (TLR2). Here, we specifically examine the lipid signatures of four species of gut-associated Bacteroides. In total we identify 170 different lipids, and we report that the range and diversity of Bacteroides lipids is species specific. Multivariate analysis reveals that the differences in the lipid signatures are largely driven by the presence and absence of plasmalogens, glycerophosphoinositols and certain SP. Moreover, we show that, in B. thetaiotaomicron, mutations altering either SP or glycine lipid biosynthesis result in significant changes in the levels of other lipids, suggesting the existence of a compensatory mechanisms required to maintain the functionality of the bacterial membrane.

The lab-on-a-chip concept, enabled by microfluidic technology, promises the integration of multiple discrete laboratory techniques into a miniaturised system. Research into microfluidics has generally focused on the development of individual elements of the total system (often with relatively limited functionality), without full consideration for integration into a complete fully optimised and miniaturised system. Typically, the operation of many of the reported lab-on-a-chip devices is dependent on the support of a laboratory framework. In this paper, a demonstrator platform for routine laboratory analysis is designed and built, which fully integrates a number of technologies into a single device with multiple domains such as fluidics, electronics, pneumatics, hydraulics, and photonics. This facilitates the delivery of breakthroughs in research, by incorporating all physical requirements into a single device. To highlight this proposed approach, this demonstrator microsystem acts as a fully integrated biochemical assay reaction system. The resulting design determines enzyme kinetics in an automated process and combines reservoirs, three-dimensional fluidic channels, optical sensing, and electronics in a low-cost, low-power and portable package.

Abstract Background Burden is well documented among carers of stroke survivors, yet current evidence is insufficient to determine if any strategies reduce this negative outcome. Existing interventions for carers of stroke survivors typically involve supporting carers according to their individual needs through face-to-face interactions and provision of information including workbooks or educational guides. To date, no interventions have been developed using a method which systematically incorporates evidence, behaviour change theories, and stakeholder involvement to change the behaviours of carers and relevant individuals who support carers. This study aimed to develop a programme plan for a theory and evidence-based intervention to reduce burden in carers of stroke survivors. Methods Informed by evidence from two systematic reviews and 33 qualitative interviews, the first four stages of Intervention Mapping were used to guide the intervention development process: 1) needs assessment; 2) identifying outcomes and objectives; 3) selecting theoretical methods and practical applications; and 4) creating a programme plan. Structured and facilitated involvement from stakeholders, including carers, researchers, and professionals from health and community services was integral to the intervention development process. Stakeholders helped to prioritise the focus of the intervention, develop the goals, outcomes and objectives for the programme, and generate and refine intervention ideas. Results Stakeholders prioritised the need for carers to feel prepared before and during the transition from hospital to home as key to reducing burden. The proposed intervention ‘Preparing is Caring’ targets this need and involves providing and signposting carers to relevant information and support for practical and emotional needs. This is to be delivered before, during, and immediately after the stroke survivor’s transition from hospital to home by a person taking on a single point of contact role. It is comprised of multiple theory-based components including: training packages for information and support providers working with carers and wider staff teams, plus elements to support carers to feel prepared. Conclusions We have developed a comprehensive programme plan for a multiple-component, theory and evidence informed behaviour change intervention aimed at preparing carers before and during the transition from hospital to home. Future work is required to refine, implement and evaluate the Preparing is Caring intervention.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Objective To review and synthesise qualitative literature relating to the views, perceptions and experiences of patients with acquired neurological conditions and their caregivers about the process of receiving information about recovery; as well as the views and experiences of healthcare professionals involved in delivering this information.Design Systematic review of qualitative studies.Data sources MEDLINE, Embase, AMED, CINAHL, PsycINFO, Web of Science and the Cochrane library were searched from their inception to July 2019.Data extraction and synthesis Two reviewers extracted data from the included studies and assessed quality using an established tool. Thematic synthesis was used to synthesise the findings of included studies.Results Searches yielded 9105 titles, with 145 retained for full-text screening. Twenty-eight studies (30 papers) from eight countries were included. Inductive analysis resulted in 11 descriptive themes, from which 5 analytical themes were generated: the right information at the right time; managing expectations; it’s not what you say, it’s how you say it; learning how to talk about recovery and manage emotions; the context of uncertainty.Conclusions Our findings highlight the inherent challenges in talking about recovery in an emotional context, where breaking bad news is a key feature. Future interventions should focus on preparing staff to meet patients’ and families’ information needs, as well as ensuring they have the skills to discuss potential recovery and break bad news compassionately and share the uncertain trajectory characteristic of acquired neurological conditions. An agreed team-based approach to talking about recovery is recommended to ensure consistency and improve the experiences of patients and their families.

Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

We demonstrate, for the first time, that a quantum flux parametron (QFP) is capable of acting as both isolator and amplifier in the readout circuit of a capacitively shunted flux qubit (CSFQ). By treating the QFP like a tunable coupler and biasing it such that the coupling is off, we show that T_{1} of the CSFQ is not impacted by Purcell loss from its low-Q readout resonator (Q_{e}=760) despite being detuned by only 40 MHz. When annealed, the QFP amplifies the qubit’s persistent current signal such that it generates a flux qubit-state-dependent frequency shift of 85 MHz in the readout resonator, which is over 9 times its linewidth. The device is shown to read out a flux qubit in the persistent current basis with fidelities surpassing 98.6% with only 80 ns integration, and reaches fidelities of 99.6% when integrated for 1 μs. This combination of speed and isolation is critical to the readout of high-coherence quantum annealers.

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex.

Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25 °C, and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.

Phenotypic heterogeneity in microbial communities enables genetically identical organisms to behave differently even under the same environmental conditions. Photorhabdus luminescens, a bioluminescent Gram-negative bacterium, contains a complex life cycle, which involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic cell types, designated as the primary (1°) and secondary (2°) cells. The 1° cells are bioluminescent, pigmented and can support nematode growth and development. Individual 1° cells undergo phenotypic switching after prolonged cultivation and convert to 2° cells, which lack the 1° specific phenotypes. The LysR-type regulator HexA has been described as major regulator of this switching process. Here we show that HexA controls phenotypic heterogeneity in a versatile way, directly and indirectly. Expression of hexA is enhanced in 2° cells, and the corresponding regulator inhibits 1° specific traits in 2° cells. HexA does not directly affect bioluminescence, a predominant 1° specific phenotype. Since the respective luxCDABE operon is repressed at the post-transcriptional level and transcriptional levels of the RNA chaperone gene hfq are also enhanced in 2° cells, small regulatory RNAs are presumably involved that are under control of HexA. Another phenotypic trait that is specific for 1° cells is quorum sensing mediated cell clumping. The corresponding pcfABCDEF operon could be identified as the first direct target of HexA, since the regulator binds to the pcfA promoter region and thereby blocks expression of the target operon. In summary, our data show that HexA fulfills the task as repressor of 1° specific features in 2° cells in a versatile way and gives first insights into the complexity of regulating phenotypic heterogeneity in Photorhabdus bacteria.

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.

The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.

Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σ(S) and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.

In the title compound, C45H40N4O5, the cyclohexane entity on the (3-cyano-2,5-dihydrofuran-2-ylidene)propanedinitrile group, which replaces the usual dimethyl substituents, has not perturbed the delocalization geometry significantly. Weak intermolecular interactions, viz. C—H...N(cyano), C—H...O(ether), C—H...π and π–π [between the aromatic rings with the shortest centroid–centroid distance of 3.603 (3) Å], consolidate the crystal packing, which exhibits voids of 57 Å3.

Carrying out fault-tolerant topological quantum computation using non-Abelian anyons (e.g., Majorana zero modes) is currently an important goal of worldwide experimental efforts. However, the Gottesman-Knill theorem [1] holds that if a system can only perform a certain subset of available quantum operations (i.e., operations from the Clifford group) in addition to the preparation and detection of qubit states in the computational basis, then that system is insufficient for universal quantum computation. Indeed, any measurement results in such a system could be reproduced within a local hidden variable theory, so there is no need for a quantum-mechanical explanation and therefore no possibility of quantum speedup [2]. Unfortunately, Clifford operations are precisely the ones available through braiding and measurement in systems supporting non-Abelian Majorana zero modes, which are otherwise an excellent candidate for topologically protected quantum computation. In order to move beyond the classically simulable subspace, an additional phase gate is required. This phase gate allows the system to violate the Bell-like Clauser-Horne-Shimony-Holt (CHSH) inequality that would constrain a local hidden variable theory. In this article, we introduce a new type of phase gate for the already-existing semiconductor-based Majorana wire systems and demonstrate how this phase gate may be benchmarked using CHSH measurements. We present an experimentally feasible schematic for such an experiment using a “measurement-only” approach that bypasses the need for explicit Majorana braiding. This approach may be scaled beyond the two-qubit system necessary for CHSH violations, leading to a well-defined platform for universal fault-tolerant quantum computation using Majorana zero modes.

Non-Abelian anyons promise to reveal spectacular features of quantum mechanics that could ultimately provide the foundation for a decoherence-free quantum computer. A key breakthrough in the pursuit of these exotic particles originated from Read and Green’s observation that the Moore-Read quantum Hall state and a (relatively simple) two-dimensional p+ip superconductor both support so-called Ising non-Abelian anyons. Here, we establish a similar correspondence between the Z_{3} Read-Rezayi quantum Hall state and a novel two-dimensional superconductor in which charge-2e Cooper pairs are built from fractionalized quasiparticles. In particular, both phases harbor Fibonacci anyons that—unlike Ising anyons—allow for universal topological quantum computation solely through braiding. Using a variant of Teo and Kane’s construction of non-Abelian phases from weakly coupled chains, we provide a blueprint for such a superconductor using Abelian quantum Hall states interlaced with an array of superconducting islands. Fibonacci anyons appear as neutral deconfined particles that lead to a twofold ground-state degeneracy on a torus. In contrast to a p+ip superconductor, vortices do not yield additional particle types, yet depending on nonuniversal energetics can serve as a trap for Fibonacci anyons. These results imply that one can, in principle, combine well-understood and widely available phases of matter to realize non-Abelian anyons with universal braid statistics. Numerous future directions are discussed, including speculations on alternative realizations with fewer experimental requirements.