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Oligodendrocyte progenitor cells (OPCs) are responsible for generating oligodendrocytes, the myelinating cells of the CNS. Life-long myelination is promoted by neuronal activity and is essential for neural network plasticity and learning. OPCs are known to contact synapses and it is proposed that neuronal synaptic activity in turn regulates their behavior. To examine this in the adult, we performed unilateral injection of the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of adult mice. We confirm BoNT/A cleaves SNAP-25 in the CA1 are of the hippocampus, which has been proven to block neurotransmission. Notably, BoNT/A significantly decreased OPC density and caused their shrinkage, as determined by immunolabeling for the OPC marker NG2. Furthermore, BoNT/A resulted in an overall decrease in the number of OPC processes, as well as a decrease in their lengths and branching frequency. These data indicate that synaptic activity is important for maintaining adult OPC numbers and cellular integrity, which is relevant to pathophysiological scenarios characterized by dysregulation of synaptic activity, such as age-related cognitive decline, Multiple Sclerosis and Alzheimer's disease.

Background Historically the main source of laboratory Xenopus laevis was the environment. The increase in genetically altered animals and evolving governmental constraints around using wild-caught animals for research has led to the establishment of resource centres that supply animals and reagents worldwide, such as the European Xenopus Resource Centre. In the last decade, centres were encouraged to keep animals in a “low microbial load” or “clean” state, where embryos are surface sterilized before entering the housing system; instead of the conventional, “standard” conditions where frogs and embryos are kept without prior surface treatment. Despite Xenopus laevis having been kept in captivity for almost a century, surprisingly little is known about the frogs as a holobiont and how changing the microbiome may affect resistance to disease. This study examines how the different treatment conditions, “clean” and “standard” husbandry in recirculating housing, affects the skin microbiome of tadpoles and female adults. This is particularly important when considering the potential for poor welfare caused by a change in husbandry method as animals move from resource centres to smaller research colonies. Results We found strong evidence for developmental control of the surface microbiome on Xenopus laevis; adults had extremely similar microbial communities independent of their housing, while both tadpole and environmental microbiome communities were less resilient and showed greater diversity. Conclusions Our findings suggest that the adult Xenopus laevis microbiome is controlled and selected by the host. This indicates that the surface microbiome of adult Xenopus laevis is stable and defined independently of the environment in which it is housed, suggesting that the use of clean husbandry conditions poses little risk to the skin microbiome when transferring adult frogs to research laboratories. This will have important implications for frog health applicable to Xenopus laevis research centres throughout the world.

TOKs are outwardly rectifying K+ channels in fungi with two pore-loops and eight transmembrane spans. Here, we describe the TOKs from four pathogens that cause the majority of life-threatening fungal infections in humans. These TOKs pass large currents only in the outward direction like the canonical isolate from Saccharomyces cerevisiae (ScTOK), and distinct from other K+ channels. ScTOK, AfTOK1 (Aspergillus fumigatus) and H99TOK (Cryptococcus neoformans grubii) are K+-selective and pass current above the K+ reversal potential. CaTOK (Candida albicans) and CnTOK (Cryptococcus neoformans neoformans) pass both K+ and Na+ and conduct above a reversal potential reflecting the mixed permeability of their selectivity filter. Mutations in CaTOK and ScTOK at sites homologous to those that open the internal gates in classical K+ channels are shown to produce inward TOK currents. A favored model for outward rectification is proposed whereby the reversal potential determines ion occupancy, and thus conductivity, of the selectivity filter gate that is coupled to an imperfectly restrictive internal gate, permitting the filter to sample ion concentrations on both sides of the membrane.

More than two-thirds of global biomass consists of vascular plants. A portion of the detritus they generate is carried into the oceans from land and highly productive blue carbon ecosystems—salt marshes, mangrove forests, and seagrass meadows. This large detrital input receives scant attention in current models of the global carbon cycle, though for blue carbon ecosystems, increasingly well-constrained estimates of biomass, productivity, and carbon fluxes, reviewed in this article, are now available. We show that the fate of this detritus differs markedly from that of strictly marine origin, because the former contains lignocellulose—an energy-rich polymer complex of cellulose, hemicelluloses, and lignin that is resistant to enzymatic breakdown. This complex can be depolymerized for nutritional purposes by specialized marine prokaryotes, fungi, protists, and invertebrates using enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases to release sugar monomers. The lignin component, however, is less readily depolymerized, and detritus therefore becomes lignin enriched, particularly in anoxic sediments, and forms a major carbon sink in blue carbon ecosystems. Eventual lignin breakdown releases a wide variety of small molecules that may contribute significantly to the oceanic pool of recalcitrant dissolved organic carbon. Marine carbon fluxes and sinks dependent on lignocellulosic detritus are important ecosystem services that are vulnerable to human interventions. These services must be considered when protecting blue carbon ecosystems and planning initiatives aimed at mitigating anthropogenic carbon emissions.

The aim of this paper is to clarify the assignments of X-ray photoelectron spectra of aluminium phosphate materials prepared from the reaction of phosphoric acid with three different aluminium precursors [Al(OH)3, Al(NO3)3 and AlCl3] at different annealing temperatures. The materials prepared have been studied by X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), infrared spectroscopy and high-resolution solid-state 31P NMR spectroscopy. A progressive polymerization from orthophosphate to metaphosphates is observed by XRD, ATR-FTIR and solid state 31P NMR, and on this basis the oxygen states observed in the XP spectra at 532.3 eV and 533.7 eV are assigned to P–O–Al and P–O–P environments, respectively. The presence of cyclic polyphosphates at the surface of the samples is also evident.

Highlights • Techniques for quantitative in vitro analysis of fluorogen-RNA aptamers are needed. • We have recently developed a novel method for the production of functional-RNA arrays. • A protocol for the production of fluorogen-binding RNA aptamer arrays is presented. • We assess the malachite green-binding aptamer and Spinach aptamer using RNA arrays. • We demonstrate application of RNA arrays to biosensors using a Spinach/SAM RNA array., The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.

Regulatory RNA-based interactions are critical for coordinating gene expression and are increasingly being targeted in synthetic biology, antimicrobial, and therapeutic fields. Bacterial trans-encoded small RNAs (sRNAs) regulate the translation and/or stability of mRNA targets through base-pairing interactions. These interactions are often integral to complex gene circuits which coordinate critical bacterial processes. The ability to predictably modulate these gene circuits has potential for reprogramming gene expression for synthetic biology and antibacterial purposes. Here, we present a novel pipeline for targeting such RNA-based interactions with antisense oligonucleotides (ASOs) in order to reprogram gene expression. As proof-of-concept, we selected sRNA–mRNA interactions that are central to the Vibrio cholerae quorum sensing pathway, required for V. cholerae pathogenesis, as a regulatory RNA-based interaction input. We rationally designed anti-sRNA ASOs to target the sRNAs and synthesized them as peptide nucleic acids (PNAs). Next, we devised an RNA array-based interaction assay to allow screening of the anti-sRNA ASOs in vitro. Finally, an Escherichia coli-based gene expression reporter assay was developed and used to validate anti-sRNA ASO regulatory activity in a cellular environment. The output from the pipeline was an anti-sRNA ASO that targets sRNAs to inhibit sRNA–mRNA interactions and modulate gene expression. This anti-sRNA ASO has potential for reprogramming gene expression for synthetic biology and/or antibacterial purposes. We anticipate that this pipeline will find widespread use in fields targeting RNA-based interactions as modulators of gene expression.

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.

White matter (WM) is a highly prominent feature in the human cerebrum and is comprised of bundles of myelinated axons that form the connectome of the brain. Myelin is formed by oligodendrocytes and is essential for rapid neuronal electrical communication that underlies the massive computing power of the human brain. Oligodendrocytes are generated throughout life by oligodendrocyte precursor cells (OPCs), which are identified by expression of the chondroitin sulphate proteoglycan NG2 (Cspg4), and are often termed NG2-glia. Adult NG2+ OPCs are slowly proliferating cells that have the stem cell–like property of self-renewal and differentiation into a pool of ‘late OPCs’ or ‘differentiation committed’ OPCs(COPs) identified by specific expression of the G-protein-coupled receptor GPR17, which are capable of differentiation into myelinating oligodendrocytes. In the adult brain, these reservoirs of OPCs and COPs ensure rapid myelination of new neuronal connections formed in response to neuronal signalling, which underpins learning and cognitive function. However, there is an age-related decline in myelination that is associated with a loss of neuronal function and cognitive decline. The underlying causes of myelin loss in ageing are manifold, but a key factor is the decay in OPC ‘stemness’ and a decline in their replenishment of COPs, which results in the ultimate failure of myelin regeneration. These changes in ageing OPCs are underpinned by dysregulation of neuronal signalling and OPC metabolic function. Here, we highlight the role of purine signalling in regulating OPC self-renewal and the potential importance of GPR17 and the P2X7 receptor subtype in age-related changes in OPC metabolism. Moreover, age is the main factor in the failure of myelination in chronic multiple sclerosis and myelin loss in Alzheimer’s disease, hence understanding the importance of purine signalling in OPC regeneration and myelination is critical for developing new strategies for promoting repair in age-dependent neuropathology.

Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

A novel and rapid approach to characterise the occurrence of contaminants of emerging concern (CECs) in river water is presented using multi-residue targeted analysis and machine learning-assisted in silico suspect screening of passive sampler extracts. Passive samplers (Chemcatcher®) configured with hydrophilic-lipophilic balance (HLB) sorbents were deployed in the Central London region of the tidal River Thames (UK) catchment in winter and summer campaigns in 2018 and 2019. Extracts were analysed by; (a) a rapid 5.5-min direct injection targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for 164 CECs and (b) a full-scan LC coupled to quadrupole time of flight mass spectrometry (QTOF-MS) method using data-independent acquisition over 15 min. From targeted analysis of grab water samples, a total of 33 pharmaceuticals, illicit drugs, drug metabolites, personal care products and pesticides (including several EU Watch-List chemicals) were identified, and mean concentrations determined at 40 ± 37 ng/L-1. For targeted analysis of passive sampler extracts, 65 unique compounds were detected with differences observed between summer and winter campaigns. For suspect screening, 59 additional compounds were shortlisted based on mass spectral database matching, followed by machine learning-assisted retention time prediction. Many of these included additional pharmaceuticals and pesticides, but also new metabolites and industrial chemicals. The novelty in this approach lies in the convenience of using passive samplers together with machine learning-assisted chemical analysis methods for rapid, time-integrated catchment monitoring of CECs.

Myelin disruption is a feature of natural aging and of Alzheimer’s disease (AD). In the CNS, myelin is produced by oligodendrocytes, which are generated throughout life by oligodendrocyte progenitor cells (OPCs). Here, we examined age-related changes in OPCs in APP/PS1 mice, a model for AD-like pathology, compared with non-transgenic (Tg) age-matched controls. Analysis was performed in the CA1 area of the hippocampus following immunolabelling for NG2 with the nuclear dye Hoescht, to identify OPC and OPC sister cells, a measure of OPC replication, together with Gpr17 and Olig2 for oligodendrocytes and myelin basic protein (MBP) immunostaining as a measure of myelination. The results indicate a decrease in the number of OPCs between 9 and 14 months in natural ageing and this occurred earlier at 9 months in APP/PS1 mice, without further decline at 14 months. The number of OPC sister cells was unaltered in natural aging, but declined significantly at 14-months in APP/PS1 mice. The number of GPR17+ and Olig2+ oligodendrocytes was not altered in APP/PS1, whereas MBP immunostaining increased between 9 and 14 months in natural ageing, but not in APP/PS1 mice. Notably, OPCs displayed marked morphological changes at 14 months in APP/PS1 mice, characterized by an overall shrinkage of OPC process domains and increased process branching, characteristic of reactive pathological changes. The results indicate that OPC and myelin disruption are pathological signs in the APP/PS1 mouse model of AD.

Significance Deconstruction of recalcitrant polymers, such as cellulose or chitin, is accomplished in nature by synergistic enzyme cocktails that evolved over millions of years. In these systems, soluble dimeric or oligomeric intermediates are typically released via interfacial biocatalysis, and additional enzymes often process the soluble intermediates into monomers for microbial uptake. The recent discovery of a two-enzyme system for polyethylene terephthalate (PET) deconstruction, which employs one enzyme to convert the polymer into soluble intermediates and another enzyme to produce the constituent PET monomers (MHETase), suggests that nature may be evolving similar deconstruction strategies for synthetic plastics. This study on the characterization of the MHETase enzyme and synergy of the two-enzyme PET depolymerization system may inform enzyme cocktail-based strategies for plastics upcycling., Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Graphical abstract, Highlights • Agathisflavone enhances remyelination through modulating neuroinflammation. • Agathisflavone induces a polarization in microglia from an M1 to an M2-like phenotype. • Agathisflavone interacts with the nuclear receptors RAR, RXRα, RXRγ, ERα, and ERβ. • Remyelination effect of agathisflavone was via ER, with a greater effect of ERα., Myelin loss is the hallmark of the demyelinating disease multiple sclerosis (MS) and plays a significant role in multiple neurodegenerative diseases. A common factor in all neuropathologies is the central role of microglia, the intrinsic immune cells of the central nervous system (CNS). Microglia are activated in pathology and can have both pro- and anti-inflammatory functions. Here, we examined the effects of the flavonoid agathisflavone on microglia and remyelination in the cerebellar slice model following lysolecithin induced demyelination. Notably, agathisflavone enhances remyelination and alters microglial activation state, as determined by their morphology and cytokine profile. Furthermore, these effects of agathisflavone on remyelination and microglial activation were inhibited by blockade of estrogen receptor α. Thus, our results identify agathisflavone as a novel compound that may act via ER to regulate microglial activation and enhance remyelination and repair.

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 24 samples at a time. There is a need for a higher throughput method to reduce cost per genome. Here we present CoronaHiT, a method capable of multiplexing up to 95 small genomes on a single Nanopore flowcell, which uses transposase mediated addition of adapters and PCR based addition of symmetric barcodes. We demonstrate the method using 48 and 94 SARS-CoV-2 genomes per flowcell, amplified using the ARTIC protocol, and compare performance with Illumina and ARTIC ONT sequencing. Results demonstrate that all sequencing methods produce inaccurate genomes when the RNA extract from SARS-CoV-2 positive clinical sample has a cycle threshold (Ct) >= 32. Results from set same set of 23 samples with a broad range of Cts show that the consensus genomes have >90% coverage (GISAID criteria) for 78.2% of samples for CoronaHiT-48, 73.9% for CoronaHiT-94, 78.2% for Illumina and 73.9% for ARTIC ONT, and all have the same clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 94 SARS-CoV-2 genomes per nanopore flowcell without compromising the quality of the resulting genomes while reducing library preparation complexity and significantly reducing cost. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E., Highlights • RNase E, an essential bacterial endoribonuclease, is a potential antibacterial target. • Three novel small molecule inhibitors of RNase E are identified. • Each inhibitor is active against RNase E from E. coli, F. tularensis and A. baumannii. • Two, as non-natural compounds, are suitable lead compounds for antibiotic development. • One, a metabolite, is a potential novel regulator of RNase E.

The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporter-protein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and post-transcription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators. We use our method to directly compare the performance of a series of previously characterized synthetic post-transcriptional riboregulators, thus demonstrating its utility in the development of synthetic regulatory modules and evaluation of gene expression regulation in general.

TREK-1 channel activity is a critical regulator of neuronal, cardiac, and smooth muscle physiology and pathology. The antidepressant peptide, spadin, has been proposed to be a TREK-1-specific blocker. Here we sought to examine the mechanism of action underlying spadin inhibition of TREK-1 channels. Heterologous expression in Xenopus laevis oocytes and electrophysiological analysis using two-electrode voltage clamp in standard bath solutions was used to characterize the pharmacological profile of wild-type and mutant murine TREK-1 and TREK-2 channels using previously established human K2P activators; arachidonic acid (AA), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), BL-1249, and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and inhibitors; spadin and barium (Ba2+). Mouse TREK-1 and TREK-2 channel currents were both significantly increased by AA, BL-1249, and CDC, similar to their human homologs. Under basal conditions, both TREK-1 and TREK-2 currents were insensitive to application of spadin, but could be blocked by Ba2+. Spadin did not significantly inhibit either TREK-1 or TREK-2 currents either chemically activated by AA, BL-1249, or CDC, or structurally activated via a gating mutation. However, pre-exposure to spadin significantly perturbed the subsequent activation of TREK-1 currents by AA, but not TREK-2. Furthermore, spadin was unable to prevent activation of TREK-1 by BL-1249, CDC, or the related bioactive lipid, DHA. Spadin specifically antagonizes the activation of TREK-1 channels by AA, likely via an allosteric mechanism. Lack of intrinsic activity may explain the absence of clinical side effects during antidepressant therapy.

Inflammation and oxidative stress are common aspects of most neurodegenerative diseases in the central nervous system. In this context, microglia and astrocytes are central to mediating the balance between neuroprotective and neurodestructive mechanisms. Flavonoids have potent anti-inflammatory and antioxidant properties. Here, we have examined the anti-inflammatory and neuroprotective potential of the flavonoid agathisflavone (FAB), which is derived from the Brazilian plant Poincianella pyramidalis, in in vitro models of neuroinflammation. Cocultures of neurons/glial cells were exposed to lipopolysaccharide (LPS, 1 &micro, g/mL) or interleukin (IL)-1&beta, (10 ng/mL) for 24 h and treated with FAB (0.1 and 1 &micro, M, 24 h). FAB displayed a significant neuroprotective effect, as measured by nitric oxide (NO) production, Fluoro-Jade B (FJ-B) staining, and immunocytochemistry (ICC) for the neuronal marker &beta, tubulin and the cell death marker caspase-3, preserving neuronal soma and increasing neurite outgrowth. FAB significantly decreased the LPS-induced microglial proliferation, identified by ICC for Iba-1/bromodeoxyuridine (BrdU) and CD68 (microglia M1 profile marker). In contrast, FAB had no apparent effect on astrocytes, as determined by ICC for glial fibrillary acidic protein (GFAP). Furthermore, FAB protected against the cytodestructive and proinflammatory effects of IL-1&beta, a key cytokine that is released by activated microglia and astrocytes, and ICC showed that combined treatment of FAB with &alpha, and &beta, estrogen receptor antagonists did not affect NF-&kappa, B expression. In addition, qPCR analysis demonstrated that FAB decreased the expression of proinflammatory molecules TNF-&alpha, IL-1&beta, and connexins CCL5 and CCL2, as well as increased the expression of the regulatory molecule IL-10. Together, these findings indicate that FAB has a significant neuroprotective and anti-inflammatory effect in vitro, which may be considered as an adjuvant for the treatment of neurodegenerative diseases.

Cryogenic storage of sperm from genetically altered Xenopus improves cost effectiveness and animal welfare associated with their use in research; currently it is routine for X. tropicalis but not reliable for X. laevis. Here we compare directly the three published protocols for Xenopus sperm freeze-thaw and determine whether sperm storage temperature, method of testes maceration and delays in the freezing protocols affect successful fertilisation and embryo development in X. laevis. We conclude that the protocol is robust and that the variability observed in fertilisation rates is due to differences between individuals. We show that the embryos made from the frozen-thawed sperm are normal and that the adults they develop into are reproductively indistinguishable from others in the colony. This opens the way for using cryopreserved sperm to distribute dominant genetically altered (GA) lines, potentially saving travel-induced stress to the male frogs, reducing their numbers used and making Xenopus experiments more cost effective., Highlights • Xenopus cryopreservation is robust using an optimized method. • Success is dependent on the quality of animals from which the sperm are taken. • Frozen sperm may now be used to distribute lines and wild-type male gametes around the world.

The process of fusion arc welding of steel pipes in power generation plants induces residual stresses which may be detrimental to the integrity and endurance of plant pipelines. P91 is high-grade steel used in the construction of pipelines carrying hot steam at high pressure, conditions which cause creep during service. Welded P91 pipes are usually subjected to post-weld heat treatment (PWHT) to mitigate the magnitude of residual stresses and temper the material, hence improving its resistance to creep. In this paper, the finite element (FE) method of modelling residual stresses due to PWHT in a circumferentially butt-welded P91 pipe is presented. The PWHT hold temperature is 760°C. The paper describes the X-Ray Diffraction (XRD) and Deep-Hole Drilling (DHD) experimental techniques and how they are applied to measure residual stresses in the welded P91 pipe after PWHT. The material property data, necessary for the FE simulation of PWHT, has been obtained from stress-relaxation tests on P91 uniaxial tensile specimens at 760°C. Good agreements have been achieved between the results of the FE method and the two sets of experimentally-measured residual stresses.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min&ndash, 1/mg&ndash, 1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-&alpha, F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-&alpha, F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min&ndash, 1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.

Inward rectifying potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis in oligodendrocytes, the myelinating cells of the central nervous system (CNS). Prominent expression of Kir4.1 has been indicated in oligodendrocytes, but the extent of expression of other Kir subtypes is unclear. Here, we used qRT-PCR to determine expression of Kir channel transcripts in the mouse optic nerve, a white matter tract comprising myelinated axons and the glia that support them. A novel finding was the high relative expression of Kir7.1, comparable to that of Kir4.1, the main glial Kir channel. Significantly, Kir7.1 immunofluorescence labelling in optic nerve sections and in isolated cells was localised to oligodendrocyte somata. Kir7.1 are known as a K+ transporting channels and, using patch clamp electrophysiology and the Kir7.1 blocker VU590, we demonstrated Kir7.1 channels carry a significant proportion of the whole cell potassium conductance in oligodendrocytes isolated from mouse optic nerves. Notably, oligodendrocytes are highly susceptible to ischemia/hypoxia and this is due at least in part to disruption of ion homeostasis. A key finding of this study is that blockade of Kir7.1 with VU590 compromised oligodendrocyte cell integrity and compounds oligodendroglial loss in ischemia/hypoxia in the oxygen–glucose deprivation (OGD) model in isolated intact optic nerves. These data reveal Kir7.1 channels are molecularly and functionally expressed in oligodendrocytes and play an important role in determining oligodendrocyte survival and myelin integrity.

There is increasing evidence that myelin disruption is related to cognitive decline in Alzheimer's disease (AD). In the CNS, myelin is produced by oligodendrocytes, which are generated throughout life by adult oligodendrocyte progenitor cells (OPCs), also known as NG2-glia. To address whether alterations in myelination are related to age-dependent changes in OPCs, we analyzed NG2 and myelin basic protein (MBP) immunolabelling in the hippocampus of 3×Tg-AD mice at 6 and 24 months of age, compared with non-Tg age-matched controls. There was an age-related decrease in MBP immunostaining and OPC density, together with a decline in the number of OPC sister cells, a measure of OPC replication. Notably, the loss of myelin and OPC sister cells occurred earlier at 6 months in 3xTg-AD, suggesting accelerated aging, although there was not a concomitant decline in OPC numbers at this age, suggesting the observed changes in myelin were not a consequence of replicative exhaustion, but possibly of OPC disruption or senescence. In line with this, a key finding is that compared to age-match controls, OPC displayed marked morphological atrophy at 6 months in 3xTg-AD followed by morphological hypertrophy at 24 months, as deduced from significant changes in total cell surface area, total cell volume, somata volume and branching of main processes. Moreover, we show that hypertrophic OPCs surround and infiltrate amyloid-β (Aβ) plaques, a key pathological hallmark of AD. The results indicate that OPCs undergo complex age-related remodeling in the hippocampus of the 3xTg-AD mouse model. We conclude that OPC disruption is an early pathological sign in AD and is a potential factor in accelerated myelin loss and cognitive decline., Graphical abstract, Highlights • Life-long generation of myelin is the function of adult oligodendrocyte progenitor cells (OPCs). • Age-related loss of myelin is accelerated in the 3xTg-AD mouse model of Alzheimer's disease (AD). • OPCs are disrupted at an early stage of 3xTg-AD. • Dysregulation of OPC and myelin loss are important biomarkers for AD-like pathology.

Amburana cearensis A.C. Smith is an endemic tree from Northeastern Brazil used in folk medicine as teas, decocts and syrups for the treatment of various respiratory and inflammatory diseases, since therapeutic properties have been attributed to compounds from its stem bark and seeds. Numerous pharmacological properties of semi-purified extracts and isolated compounds from A. cearensis have been described in several biological systems, ranging from antimicrobial to anti-inflammatory effects. Some of these activities are attributed to coumarins and phenolic compounds, the major compounds present in A. cearensis seed extracts. Multiple lines of research demonstrate these compounds reduce oxidative stress, inflammation and neuronal death induced by glutamate excitotoxicity, events central to most neuropathologies, including Alzheimer’s disease (AD) and Parkinson’s Disease (PD). This review focuses on the botanical aspects, folk medicine use, biological effects and pharmacological activities of A. cearensis compounds and their potential as novel non-toxic drugs for the treatment of neurodegenerative diseases.

Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K+ transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein‐enhanced green fluorescent protein (GFAP‐EGFP], myelinating oligodendrocytes (PLP‐DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra‐creERT2/Rosa26‐YFP double‐transgenic mice) and all oligodendrocyte lineage cells (SOX10‐EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end‐feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone‐glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K+ homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.

Astrocytes are multifunctional glial cells that play essential roles in supporting synaptic signalling and white matter-associated connectivity. There is increasing evidence that astrocyte dysfunction is involved in several brain disorders, including bipolar disorder (BD), depression and schizophrenia. The mood stabiliser lithium is a frontline treatment for BD, but the mechanisms of action remain unclear. Here, we demonstrate that astrocytes are direct targets of lithium and identify unique astroglial transcriptional networks that regulate specific molecular changes in astrocytes associated with BD and schizophrenia, together with Alzheimer’s disease (AD). Using pharmacogenomic analyses, we identified novel roles for the extracellular matrix (ECM) regulatory enzyme lysyl oxidase (LOX) and peroxisome proliferator-activated receptor gamma (PPAR-γ) as profound regulators of astrocyte morphogenesis. This study unravels new pathophysiological mechanisms in astrocytes that have potential as novel biomarkers and potential therapeutic targets for regulating astroglial responses in diverse neurological disorders.

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M−1 s−1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease. Keywords: Mitochondria, ATP synthase, Fertilisation, Development, Redox signalling

Significance Lignin is an abundant but underutilized heterogeneous polymer found in terrestrial plants. In current lignocellulosic biorefinery paradigms, lignin is primarily slated for incineration, but for a nonfood plant-based bioeconomy to be successful, lignin valorization is critical. An emerging concept to valorize lignin uses aromatic–catabolic pathways and microbes to funnel heterogeneous lignin-derived aromatic compounds to single high-value products. For this approach to be viable, the discovery and engineering of enzymes to conduct key reactions is critical. In this work, we have engineered a two-component cytochrome P450 enzyme system to conduct one of the most important reactions in biological lignin conversion: aromatic O-demethylation of syringol, the base aromatic unit of S-lignin, which is highly abundant in hardwoods and grasses., Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset., Graphical abstract Unlabelled Image, Highlights • There are three apolipoprotein E isoforms, and E4E4 increases the risk for Alzheimer's disease. • ApoE2, E3 and E4 share a quaternary, tertiary and secondary structures, and stability. • ApoE4 forms non-amyloid-like filaments, while ApoE2 and E3 do not. • Assembly of E4 may play a role increased risk of late-onset Alzheimer's disease.

Two species of the clawed frog family, Xenopus laevis and X. tropicalis, are widely used as tools to investigate both normal and disease-state biochemistry, genetics, cell biology, and developmental biology. To support both frog specialist and non-specialist scientists needing access to these models for their research, a number of centralized resources exist around the world. These include centers that hold live and frozen stocks of transgenic, inbred and mutant animals and centers that hold molecular resources. This infrastructure is supported by a model organism database. Here, we describe much of this infrastructure and encourage the community to make the best use of it and to guide the resource centers in developing new lines and libraries.

Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.

Understanding the sorption of pharmaceuticals to sewage sludge during waste water treatment processes is important for understanding their environmental fate and in risk assessments. The degree of sorption is defined by the sludge/water partition coefficient (Kd). Experimental Kd values (n = 297) for active pharmaceutical ingredients (n = 148) in primary and activated sludge were collected from literature. The compounds were classified by their charge at pH 7.4 (44 uncharged, 60 positively and 28 negatively charged, and 16 zwitterions). Univariate models relating log Kd to log Kow for each charge class showed weak correlations (maximum R2 = 0.51 for positively charged) with no overall correlation for the combined dataset (R2 = 0.04). Weaker correlations were found when relating log Kd to log Dow. Three sets of molecular descriptors (Molecular Operating Environment, VolSurf and ParaSurf) encoding a range of physico-chemical properties were used to derive multivariate models using stepwise regression, partial least squares and Bayesian artificial neural networks (ANN). The best predictive performance was obtained with ANN, with R2 = 0.62–0.69 for these descriptors using the complete dataset. Use of more complex Vsurf and ParaSurf descriptors showed little improvement over Molecular Operating Environment descriptors. The most influential descriptors in the ANN models, identified by automatic relevance determination, highlighted the importance of hydrophobicity, charge and molecular shape effects in these sorbate-sorbent interactions. The heterogeneous nature of the different sewage sludges used to measure Kd limited the predictability of sorption from physico-chemical properties of the pharmaceuticals alone. Standardization of test materials for the measurement of Kd would improve comparability of data from different studies, in the long-term leading to better quality environmental risk assessments., Graphical abstract Image 1, Highlights • Understanding sorption of pharmaceuticals to sludge is important in risk assessment. • Predicting sorption on molecular properties limited by heterogeneous nature of sludge • Models based only on the partition coefficient gave poor predictive models. • Non-linear artificial neural network models improve predictability. • Descriptors that influenced sorbate-sorbent interactions were identified.

Background and Aims Bioenergy is central for the future energy mix to mitigate climate change impacts; however, its intricate link with the water cycle calls for an evaluation of the carbon–water nexus in biomass production. The great challenge is to optimize trade-offs between carbon harvest and water use by choosing cultivars that combine low water use with high productivity. Methods Regional scenarios were simulated over a range of willow genotype × environment interactions for the major UK soil × climate variations with the process-based model LUCASS. Soil available water capacity (SAWC) ranged from 51 to 251 mm and weather represented the north-west (wet, cool), north-east (dry, cool), south-west (wet, warm) and south-east (dry, warm) of the UK. Scenario simulations were evaluated for small/open narrow-leaf (NL) versus large/closed broad-leaf (BL) willow canopy phenotypes using baseline (1965–89) and warmer recent (1990–2014) weather data. Key Results The low productivity under baseline climate in the north could be compensated by choosing BL cultivars (e.g. ‘Endurance’). Recent warmer climate increased average productivity by 0.5–2.5 t ha−1, especially in the north. The modern NL cultivar ‘Resolution’ had the smallest and most efficient water use. On marginal soils (SAWC Conclusions With an increase in air temperature and occasional water scarcities expected with climate change, high-yielding NL cultivars should be the preferred choice for sustainable use of marginal lands and reduced competition with agricultural food crops.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining., Marine woodborers can digest woody biomass without the help of gut microbiota but the mechanism has remained unclear. Here, the authors provide evidence that the woodborer’s respiratory protein hemocyanin plays a central role in wood digestion and may offer a route toward biorefining of woody plant biomass.

Exposure to ambient particulate matter (PM) air pollution is a leading risk factor for morbidity and mortality, associated with up to 8.9 million deaths/year worldwide. Measurement of personal exposure to PM is hindered by poor spatial resolution of monitoring networks. Low-cost PM sensors may improve monitoring resolution in a cost-effective manner but there are doubts regarding data reliability. PM sensor boxes were constructed using four low-cost PM micro-sensor models. Three boxes were deployed at each of two schools in Southampton, UK, for around one year and sensor performance was analysed. Comparison of sensor readings with a nearby background station showed moderate to good correlation (0.61

BackgroundRhamnolipids, biosurfactants with a wide range of biomedical applications, are amphiphilic molecules produced on the surfaces of or excreted extracellularly by bacteria includingPseudomonas aeruginosa. However,Pseudomonas putidais a non-pathogenic model organism with greater metabolic versatility and potential for industrial applications.MethodsWe investigate in silico the metabolic capabilities ofP. putidafor rhamnolipids biosynthesis using statistical, metabolic and synthetic engineering approaches after introducing key genes (RhlAandRhlB) fromP. aeruginosainto a genome-scale model ofP. putida. This pipeline combines machine learning methods with multi-omic modelling, and drives the engineeredP. putidamodel toward an optimal production and export of rhamnolipids out of the membrane.ResultsWe identify a substantial increase in synthesis of rhamnolipids by the engineered model compared to the control model. We apply statistical and machine learning techniques on the metabolic reaction rates to identify distinct features on the structure of the variables and individual components driving the variation of growth and rhamnolipids production. We finally provide a computational framework for integrating multi-omics data and identifying latent pathways and genes for the production of rhamnolipids inP. putida.ConclusionsWe anticipate that our results will provide a versatile methodology for integrating multi-omics data for topological and functional analysis ofP. putidatoward maximization of biosurfactant production.

Scientific methods for assessing animal affect, especially affective valence (positivity or negativity), allow us to evaluate animal welfare and the effectiveness of 3Rs Refinements designed to improve wellbeing. Judgement bias tasks measure valence; however, task-training may be lengthy and/or require significant time from researchers. Here we develop an automated and self-initiated judgement bias task for rats which capitalises on their natural investigative behaviour. Rats insert their noses into a food trough to start trials. They then hear a tone and learn either to stay for 2 s to receive a food reward or to withdraw promptly to avoid an air-puff. Which contingency applies is signalled by two different tones. Judgement bias is measured by responses to intermediate ambiguous tones. In two experiments we show that rats learn the task in fewer sessions than other automated variants, generalise responses across ambiguous tones as expected, self-initiate 4–5 trials/min, and can be tested repeatedly. Affect manipulations generate main effect trends in the predicted directions, although not localised to ambiguous tones, so further construct validation is required. We also find that tone-reinforcer pairings and reinforcement or non-reinforcement of ambiguous trials can affect responses to ambiguity. This translatable task should facilitate more widespread uptake of judgement bias testing.

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion., Catabolizing lignin-derived aromatic compounds requires an aryl-O-demethylation step. Here the authors present the structures of GcoA and GcoB, a cytochrome P450-reductase pair that catalyzes aryl-O-demethylations and show that GcoA displays broad substrate specificity, which is of interest for biotechnology applications.

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA–small molecule and RNA–RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.

Significance Synthetic polymers are ubiquitous in the modern world but pose a global environmental problem. While plastics such as poly(ethylene terephthalate) (PET) are highly versatile, their resistance to natural degradation presents a serious, growing risk to fauna and flora, particularly in marine environments. Here, we have characterized the 3D structure of a newly discovered enzyme that can digest highly crystalline PET, the primary material used in the manufacture of single-use plastic beverage bottles, in some clothing, and in carpets. We engineer this enzyme for improved PET degradation capacity and further demonstrate that it can also degrade an important PET replacement, polyethylene-2,5-furandicarboxylate, providing new opportunities for biobased plastics recycling., Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMOPhi1) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMOPhi1. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMOPhi1 for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.

Assessment of the fate of pharmaceutical residues in the environment involves the measurement or prediction of their sewage sludge partition coefficient (Kd). Sewage sludge can be classified into four types: primary, activated, secondary and digested, each one with different physical and chemical properties. Published studies have measured Kd for pharmaceuticals in a variety of sludge types. This paper discusses the variability of reported Kd values of pharmaceuticals in different types of sewage sludge, using a dataset generated from the literature. Using a meta-analysis approach, it was shown that the measured Kd values depend on the type of sludge used in the test. Recommendations are given for the type of sludge to be used when studying the partitioning behaviour of pharmaceuticals in waste water treatment plants. Activated sludge is preferred due to its more homogenous nature and the ease of collection of consistent samples at a plant. Weak statistical relationships were found between Kd values for activated and secondary sludge, and for activated and digested sludge. Pooling of Kd values for these sludge types is not recommended for preliminary fate and risk assessments. In contrast, statistical analyses found stronger similarities between Kd values reported for the same pharmaceutical in primary and activated sludges. This allows the pooling of experimental values for these two sludge types to obtain a larger dataset for modelling purposes.

Graphical abstract, Highlights • White matter astrocytes are vulnerable to ischemia. • Immunolabelling and calcium imaging demonstrate mGluR5 in white matter astrocytes. • Activation of mGluR protects astrocytes against ischemic damage., Astrocytes perform essential neuron-supporting functions in the central nervous system (CNS) and their disruption has devastating effects on neuronal integrity in multiple neuropathologies. Although astrocytes are considered resistant to most pathological insults, ischemia can result in astrocyte injury and astrocytes in postnatal white matter are particularly vulnerable. Metabotropic glutamate receptors (mGluR) are neuroprotective in ischemia and are widely expressed by astrocytes throughout CNS grey matter, but their potential cytoprotective role in astrocytes had not been determined. Here, we identify functional expression of group I mGluR in white matter astrocytes and demonstrate their activation protects astrocytes from ischemic damage in the postnatal mouse optic nerve. Optic nerve astrocytes are shown to express mGluR5 using immunolabelling of sections and explant cultures from transgenic reporter mice in which GFAP drives expression of EGFP. In addition, using Fluo-4 calcium imaging in isolated intact optic nerves, we show that the group I/II mGluR agonist ACPD and the specific group I mGluR agonist DHPG evoke glial Ca2+ signals that were significantly inhibited by the group I mGluR antagonist AIDA. A key finding is that activation of group I mGluR protects astrocytes against oxygen-glucose deprivation (OGD) in situ, in isolated intact optic nerves from GFAP-EGFP mice. This study identifies a role for group I mGluR in protecting astrocytes against ischemia in postnatal white matter and suggests this may be a strategy for limiting damage in neuropathologies involving excitotoxity.

Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source on the Planet. Turnover of woody biomass is a critical component of the planetary carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon recycling by reprocessing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal’s gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatusis mostly comprised of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that accounts for more than half of the total protein content in the cecum and functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.

Air Quality (AQ) is a very topical issue for many cities and has a direct impact on the health of its citizens. We propose to investigate the air quality of a large UK city using low-cost Particulate Matter (PM) micro-sensors, and compare them with government operated air quality stations. In this pilot deployment we design and build six AQ IoT devices, each with four different low-cost PM sensors and deploy them at two locations within the city. These devices are equipped with LoRaWAN wireless network transceivers to test city scale Low-Power Wide-Area Network network coverage. We conclude that some low-cost PM sensors are viable for monitoring AQ and demonstrate that our device design can be used via LoRaWAN to facilitate more granular city coverage without limitations of network access. Based on these findings we intend to deploy a larger LoRaWAN enabled Air Quality sensor network deployment across the city.

Highlights • GABAA receptors (GABAARs) curtail stress-induced activation of the HPA axis. • Stressful challenges evoke de novo brain synthesis of GABAAR-active neurosteroids (NS). • NS inhibit the output of CRF-releasing neurones of the hypothalamus. • NS actions in the hypothalamus are blunted in rodent models of early-life adversity. • NS may be important molecular messengers in the programming of the stress-response., Regulation of hypothalamic–pituitary–adrenocortical (HPA) axis activity by stress is a fundamental survival mechanism and HPA-dysfunction is implicated in psychiatric disorders. Adverse early life experiences, e.g. poor maternal care, negatively influence brain development and programs an abnormal stress response by encoding long-lasting molecular changes, which may extend to the next generation. How HPA-dysfunction leads to the development of affective disorders is complex, but may involve GABAA receptors (GABAARs), as they curtail stress-induced HPA axis activation. Of particular interest are endogenous neurosteroids that potently modulate the function of GABAARs and exhibit stress-protective properties. Importantly, neurosteroid levels rise rapidly during acute stress, are perturbed in chronic stress and are implicated in the behavioural changes associated with early-life adversity. We will appraise how GABAAR-active neurosteroids may impact on HPA axis development and the orchestration of the stress-evoked response. The significance of these actions will be discussed in the context of stress-associated mood disorders.

The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers., In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.