Multiple toxins and a protease contribute to the aphid-killing ability of Pseudomonas fluorescens PpR24.

Aphids are globally important pests causing damage to a broad range of crops. Due to insecticide resistance, there is an urgent need to develop alternative control strategies. In our previous work, we found Pseudomonas fluorescens PpR24 can orally infect and kill the insecticide-resistant green-peach aphid (Myzus persicae). However, the genetic basis of the insecticidal capability of PpR24 remains unclear. Genome sequencing of PpR24 confirmed the presence of various insecticidal toxins such as Tc (toxin complexes), Rhs (rearrangement hotspot) elements, and other insect-killing proteases. Upon aphids infection with PpR24, RNA-Seq analysis revealed 193 aphid genes were differentially expressed with down-regulation of 16 detoxification genes. In addition, 1325 PpR24 genes (542 were upregulated and 783 downregulated) were subject to differential expression, including genes responsible for secondary metabolite biosynthesis, the iron-restriction response, oxidative stress resistance, and virulence factors. Single and double deletion of candidate virulence genes encoding a secreted protease (AprX) and four toxin components (two TcA-like; one TcB-like; one TcC-like insecticidal toxins) showed that all five genes contribute significantly to aphid killing, particularly AprX. This comprehensive host-pathogen transcriptomic analysis provides novel insight into the molecular basis of bacteria-mediated aphid mortality and the potential of PpR24 as an effective biocontrol agent.

A review of the effect of iron supplementation on the gut microbiota of children in developing countries and the impact of prebiotics.

Iron is essential for many physiological functions of the body, and it is required for normal growth and development. Iron deficiency (ID) is the most common form of micronutrient malnutrition and is particularly prevalent in infants and young children in developing countries. Iron supplementation is considered the most effective strategy to combat the risk of ID and ID anaemia (IDA) in infants, although iron supplements cause a range of deleterious gut-related problems in malnourished children. The purpose of this review is to assess the available evidence on the effect of iron supplementation on the gut microbiota during childhood ID and to further assess whether prebiotics offer any benefits for iron supplementation. Prebiotics are well known to improve gut-microbial health in children, and recent reports indicate that prebiotics can mitigate the adverse gut-related effects of iron supplementation in children with ID and IDA. Thus, provision of prebiotics alongside iron supplements has the potential for an enhanced strategy for combatting ID and IDA among children in the developing world. However, further understanding is required before the benefit of such combined treatments of ID in nutritionally deprived children across populations can be fully confirmed. Such enhanced understanding is of high relevance in resource-poor countries where ID, poor sanitation and hygiene, alongside inadequate access to good drinking water and poor health systems, are serious public health concerns.

A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

LIPID MAPS: update to databases and tools for the lipidomics community.

LIPID MAPS (LIPID Metabolites and Pathways Strategy), www.lipidmaps.org, provides a systematic and standardized approach to organizing lipid structural and biochemical data. Founded 20 years ago, the LIPID MAPS nomenclature and classification has become the accepted community standard. LIPID MAPS provides databases for cataloging and identifying lipids at varying levels of characterization in addition to numerous software tools and educational resources, and became an ELIXIR-UK data resource in 2020. This paper describes the expansion of existing databases in LIPID MAPS, including richer metadata with literature provenance, taxonomic data and improved interoperability to facilitate FAIR compliance. A joint project funded by ELIXIR-UK, in collaboration with WikiPathways, curates and hosts pathway data, and annotates lipids in the context of their biochemical pathways. Updated features of the search infrastructure are described along with implementation of programmatic access via API and SPARQL. New lipid-specific databases have been developed and provision of lipidomics tools to the community has been updated. Training and engagement have been expanded with webinars, podcasts and an online training school.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that Padi4 expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Functions and mechanisms of the GPCR adaptor protein Norbin.

Norbin (Neurochondrin, NCDN) is a highly conserved 79 kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.

Evidence-based consensus guidelines for the management of catatonia: Recommendations from the British Association for Psychopharmacology.

The British Association for Psychopharmacology developed an evidence-based consensus guideline on the management of catatonia. A group of international experts from a wide range of disciplines was assembled. Evidence was gathered from existing systematic reviews and the primary literature. Recommendations were made on the basis of this evidence and were graded in terms of their strength. The guideline initially covers the diagnosis, aetiology, clinical features and descriptive epidemiology of catatonia. Clinical assessments, including history, physical examination and investigations are then considered. Treatment with benzodiazepines, electroconvulsive therapy and other pharmacological and neuromodulatory therapies is covered. Special regard is given to periodic catatonia, malignant catatonia, neuroleptic malignant syndrome and antipsychotic-induced catatonia. There is attention to the needs of particular groups, namely children and adolescents, older adults, women in the perinatal period, people with autism spectrum disorder and those with certain medical conditions. Clinical trials were uncommon, and the recommendations in this guideline are mainly informed by small observational studies, case series and case reports, which highlights the need for randomised controlled trials and prospective cohort studies in this area.

Impact of inorganic iron and haem on the human gut microbiota; An in vitro batch-culture approach.

Although iron is an essential nutrient for humans, as well as for almost all other organisms, it is poorly absorbed (~15%) from the diet such that most passes through the upper gut into the large intestine. The colonic microbiota is thus exposed to, and potentially influenced by, such residual iron which could have an impact on human health. The aim of the research described here is to determine how the major forms of dietary iron (inorganic iron and haem) influence metabolic activity and composition of the human gut microbiota by utilizing an in vitro parallel, pH-controlled anaerobic batch culture approach. Controlled iron provision was enabled by the design of a 'modified' low-iron gut-model medium whereby background iron content was reduced from 28 to 5 µM. Thus, the impact of both low and high levels of inorganic and haem iron (18-180 µM and 7.7-77 µM, respectively) could be explored. Gut-microbiota composition was determined using next generation sequencing (NGS) based community profiling (16S rRNA gene sequencing) and flow-fluorescent in situ hybridization (FISH). Metabolic-end products (organic acids) were quantified using gas chromatography (GC) and iron incorporation was estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that differences in iron regime induced significant changes in microbiota composition when low (0.1% w/v) fecal inoculation levels were employed. An increase in haem levels from 7.7 to 77 µM (standard levels employed in gut culture studies) resulted in reduced microbial diversity, a significant increase in Enterobacteriaceae and lower short chain fatty acid (SCFA) production. These effects were countered when 18 µM inorganic iron was also included into the growth medium. The results therefore suggest that high-dietary haem may have a detrimental effect on health since the resulting changes in microbiota composition and SCFA production are indicators of an unhealthy gut. The results also demonstrate that employing a low inoculum together with a low-iron gut-model medium facilitated in vitro investigation of the relationship between iron and the gut microbiota.

Staphylococcus aureus FadB is a dehydrogenase that mediates cholate resistance and survival under human colonic conditions.

Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host's innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon.

Multi-omics analyses demonstrate a critical role for EHMT1 methyltransferase in transcriptional repression during oogenesis.

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2 cKO, Ehmt1 cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2 cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)-based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.

Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B.

DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.

Guiding the choice of informatics software and tools for lipidomics research applications.

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

Native Size-Exclusion Chromatography-Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes.

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Effect of Superovulation Treatment on Oocyte's DNA Methylation.

Controlled ovarian stimulation is a necessary step in some assisted reproductive procedures allowing a higher collection of female gametes. However, consequences of this stimulation for the gamete or the offspring have been shown in several mammals. Most studies used comparisons between oocytes from different donors, which may contribute to different responses. In this work, we use the bovine model in which each animal serves as its own control. DNA methylation profiles were obtained by single-cell whole-genome bisulfite sequencing of oocytes from pre-ovulatory unstimulated follicles compared to oocytes from stimulated follicles. Results show that the global percentage of methylation was similar between groups, but the percentage of methylation was lower for non-stimulated oocytes in the imprinted genes APEG3, MEG3, and MEG9 and higher in TSSC4 when compared to stimulated oocytes. Differences were also found in CGI of imprinted genes: higher methylation was found among non-stimulated oocytes in MEST (PEG1), IGF2R, GNAS (SCG6), KvDMR1 ICR UMD, and IGF2. In another region around IGF2, the methylation percentage was lower for non-stimulated oocytes when compared to stimulated oocytes. Data drawn from this study might help to understand the molecular reasons for the appearance of certain syndromes in assisted reproductive technologies-derived offspring.

Effects of iron deficiency and iron supplementation at the host-microbiota interface: Could a piglet model unravel complexities of the underlying mechanisms?

Iron deficiency is the most prevalent human micronutrient deficiency, disrupting the physiological development of millions of infants and children. Oral iron supplementation is used to address iron-deficiency anemia and reduce associated stunting but can promote infection risk since restriction of iron availability serves as an innate immune mechanism against invading pathogens. Raised iron availability is associated with an increase in enteric pathogens, especially Enterobacteriaceae species, accompanied by reductions in beneficial bacteria such as Bifidobacteria and lactobacilli and may skew the pattern of gut microbiota development. Since the gut microbiota is the primary driver of immune development, deviations from normal patterns of bacterial succession in early life can have long-term implications for immune functionality. There is a paucity of knowledge regarding how both iron deficiency and luminal iron availability affect gut microbiota development, or the subsequent impact on immunity, which are likely to be contributors to the increased risk of infection. Piglets are naturally iron deficient. This is largely due to their low iron endowments at birth (primarily due to large litter sizes), and their rapid growth combined with the low iron levels in sow milk. Thus, piglets consistently become iron deficient within days of birth which rapidly progresses to anemia in the absence of iron supplementation. Moreover, like humans, pigs are omnivorous and share many characteristics of human gut physiology, microbiota and immunity. In addition, their precocial nature permits early maternal separation, individual housing, and tight control of nutritional intake. Here, we highlight the advantages of piglets as valuable and highly relevant models for human infants in promoting understanding of how early iron status impacts physiological development. We also indicate how piglets offer potential to unravel the complexities of microbiota-immune responses during iron deficiency and in response to iron supplementation, and the link between these and increased risk of infectious disease.

Librarian: A quality control tool to analyse sequencing library compositions.

BACKGROUND: Robust analysis of DNA sequencing data needs to include a set of quality control steps to ensure that technical bias is kept to a minimum. A metric easily obtained is the frequency of each of the nucleobases for each position across all sequencing reads. Here, we explore the differences in nucleobase compositions of various library types produced by standard experimental methodologies.  Methods: We obtained the compositions of nearly 3000 publicly available datasets and subjected them to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction for a two-dimensional representation of their composition characteristics.   Results: We find that most library types result in a specific composition profile. We use this to give an estimate of how strongly the composition of a test library resembles the profiles of previously published libraries, and how likely the test sample is to be of a particular type. We introduce Librarian, a user-friendly web application and command line tool which enables checking base compositions of test libraries against known library types.   Conclusions: Library preparation methods strongly influence the per position nucleobase content. By comparing test libraries to a database of previously published library types we can make predictions regarding the library preparation method. Librarian is a user-friendly tool to access this information for quality assurance purposes as discrepancies can flag potential irregularities very early on.

Miniaturised broth microdilution for simplified antibiotic susceptibility testing of Gram negative clinical isolates using microcapillary devices.

Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical agreement (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement with existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST.