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Membrane-permeable trehalose 6-phosphate precursor spray increases wheat yields in field trials.
Trehalose 6-phosphate (T6P) is an endogenous sugar signal in plants that promotes growth, yet it cannot be introduced directly into crops or fully genetically controlled. Here we show that wheat yields were improved using a timed microdose of a plant-permeable, sunlight-activated T6P signaling precursor, DMNB-T6P, under a variety of agricultural conditions. Under both well-watered and water-stressed conditions over 4 years, DMNB-T6P stimulated yield of three elite varieties. Yield increases were an order of magnitude larger than average annual genetic gains of breeding programs and occurred without additional water or fertilizer. Mechanistic analyses reveal that these benefits arise from increased CO2 fixation and linear electron flow ('source') as well as from increased starchy endosperm volume, enhanced grain sieve tube development and upregulation of genes for starch, amino acid and protein synthesis ('sink'). These data demonstrate a step-change, scalable technology with net benefit to the environment that could provide sustainable yield improvements of diverse staple cereal crops.
Repeated administration of L-alanine to mice reduces behavioural despair and increases hippocampal mammalian target of rapamycin signalling: Analysis of gender and metabolic effects.
BACKGROUND: The amino acid L-alanine, has been shown to be elevated in biofluids during major depression but its relevance remains unexplored. AIM: We have investigated the effects of repeated L-alanine administration on emotional behaviours and central gene expression in mice. METHODS: Mice received a daily, 2-week intraperitoneal injection of either saline or L-alanine at 100 or 200 mg/kg and were exposed to the open field, light-dark box and forced swim test. The expression of L-alanine transporters (asc-1, ASCT2), glycine receptor subunits (GlyRs), NMDA receptor subunits (GluNs) mRNAs were measured, together with western blots of the signalling protein mammalian target of rapamycin (mTOR). Since L-alanine modulates glucose homeostasis, peripheral and central metabolomes were evaluated with 1H-NMR. RESULTS: L-alanine administration at 100 mg/kg, but not at 200 mg/kg, to both male and female mice increased latency to float and reduced floating time in the forced swim test, but had no effect on anxious behaviour in the open field and light-dark box tests. There was a significant reduction in mRNAs encoding asc-1 and ASCT2 and GluN2B in the hippocampus of mice following 100 mg/kg L-alanine only. On western blots, hippocampal GluN2B immunoreactivity was reduced, but mTOR signalling was increased in the 100 mg/kg L-alanine group. 1H-NMR revealed gender-specific changes in the forebrain, plasma and liver metabolomes only at 200 mg/kg of L-alanine. CONCLUSIONS: Our data suggest that L-alanine may have antidepressant-like effect that may involve the modulation of glutamate neurotransmission independently of metabolism. In major depression, therefore, elevated L-alanine may be a homeostatic response to pathophysiological processes, though this will require further investigation.
Myelopoiesis is temporally dynamic and is regulated by lifestyle to modify multiple sclerosis.
Monocytes and neutrophils from the myeloid lineage contribute to multiple sclerosis (MS), but the dynamics of myelopoiesis during MS are unclear. Here we uncover a disease stage-specific relationship between lifestyle, myelopoiesis and neuroinflammation. In mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), myelopoiesis in the femur, vertebrae and spleen is elevated prior to disease onset and during remission, preceding the peaks of clinical disability and neuroinflammation. In progressive EAE (P-EAE), vertebral myelopoiesis rises steadily throughout disease, while femur and splenic myelopoiesis is elevated early before waning later during disease height. In parallel, sleep disruption or hyperlipidemia and cardiometabolic syndrome augment M-CSF generation and multi-organ myelopoiesis to worsen P-EAE clinical symptoms, neuroinflammation, and spinal cord demyelination, with M-CSF blockade abrogating these symptoms. Lastly, results from a previous trial show that Mediterranean diet restrains myelopoietic activity and myeloid lineage progenitor skewing and improves clinical symptomology of MS. Together, our data suggest that myelopoiesis in MS is dynamic and dependent on disease stage and location, and that lifestyle factors modulate disease by influencing M-CSF-mediated myelopoiesis.
Permissive central tolerance plus defective peripheral checkpoints license pathogenic memory B cells in CASPR2-antibody encephalitis.
Autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the development of disease-causing B cells and autoantibodies. Convention suggests that such autoreactivities are generated during germinal center reactions. Here, we explore earlier immune checkpoints, focusing on patients with contactin-associated protein-like 2 (CASPR2)-autoantibody encephalitis. In both disease and health, high (~0.5%) frequencies of unmutated CASPR2-reactive naïve B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with pathogenic effects in neuronal cultures and mice. The unmutated, precursor memory B cell receptors showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results identify permissive central tolerance, defective peripheral tolerance, and autoantigen-specific tolerance thresholds in humans as sequential steps that license CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches, with an experimental paradigm applicable across autoimmunity.
The modernized classification of cardiac anti-arrhythmic drugs: its application to clinical practice.
Cardiac arrhythmias pose a major public health problem and pharmacological intervention remains key to their therapy. The landmark Vaughan Williams (VW, 1970) classification utilizing known actions of then available anti-arrhythmic drugs (AADs) became and remains central to management, but requires revision in response to extensive subsequent advances. Our modernized antiarrhythmic drug (AAD) classification reflected and sought to facilitate such fundamental physiological and clinical development. We here respond to requests for an adaptation of our scheme specifically focussed at clinical practice. This adaptation: (1) improves accessibility of our original scheme to clinical practice, focussing on key AADs in clinical use rather than investigational new drugs (INDs) whilst still conserving and encompassing the classic VW scheme. We nevertheless (2) preserve a rational conceptual framework based on current understanding of the relevant electrophysiological events, their underlying cellular or molecular cardiomyocyte targets and the functional mechanisms they mediate. Additionally, (3) the adopted subclasses within each AAD class parallel clinical practice in including only subclasses containing established AADs, or approved potential off-label drugs, as opposed to those only including INDs. Finally, (4) the simplified scheme remains flexible, permitting drugs to be placed in multiple classes where required, and the future addition of classes and subclasses in the light of future investigations and clinical approvals. We thus derive from our comprehensive modernized AAD classification a more focussed and simpler scheme, for clinical use. This both modernizes but preserves the classic Vaughan Williams classification, and remains flexible accommodating for future developments.
Physiological Function of Cyclic Nucleotide Phosphodiesterases in Atrial Myocytes and their Potential as Targets in Atrial Fibrillation.
Cyclic nucleotide hy drolysing phosphodiesterases (PDEs) are key regulators of cyclic nucleotide (e.g. cAMP and cGMP) signalling. Here we examine the role of PDEs in the physiology of atrial myocytes (AMs), the pathogenesis of atrial fibrillation (AF) and the potential of PDEs as therapeutic targets for AF. PDE1-5 and 8 are present and functional in AMs. PDE2-4 are important regulators of AM contraction but their role beyond atrial contractility is unclear. The role of PDE1,5 and 8 in healthy AMs is unknown but of interest because of their roles in ventricular myocytes. We propose that PDE2-5 and PDE8 are potential targets to prevent the triggering of AF considering their effects on Ca2+ handling and /or electrical activity. PDE1-5 are possible targets to treat patients with paroxysmal or persistent AF caused by pulmonary vein automaticity. PDE8B2 is a possible target for patients with persistent AF due to its altered expression. Research should aim to identify the presence, localisation, and function of specific PDE isoforms in human atria. Ultimately, the paucity of PDE isoform-specific small molecule modulators and the difficulty of delivering PDE-targeted medications or therapies to particular cell types limit current research and its application.
Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.
Development of a fluorescence-based assay for RecBCD activity using Functional Data Analysis and Design of Experiments
Biochemical assays are essential tools in biological research and drug discovery, but optimisation of these assays is often a challenging and lengthy process due to the wide range of input...
Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide.
The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.
Protocol: A metabolomic analysis of convalescent inflammatory conditions
Background ‘The term ‘long covid’ describes persistent symptoms following infection with SARS-CoV-2 that are not explained by an alternative diagnosis. It embraces a number of globally used terms and reported prevalence is highly variable. In the United Kingdom (UK) in 2023, approximately 2.9% of the population were thought to be affected. The condition manifests in a constellation of fluctuant symptoms, which persist beyond the acute infection and frequently profoundly impact an individual’s functional and relational capacity. The underlying mechanisms remain imperfectly understood and there is great demand for diagnostic tools that distinguish long covid from other chronic conditions. This study aims to utilise metabolomics to develop such a test and identify potential pathophysiological mechanisms. Methods Blood and urine samples will be collected at two timepoints at least 9 months apart from non-hospitalised individuals with a previous confirmed COVID-19 infection. This population will be divided into those who recovered completely within six weeks and those who continue to experience persistent symptoms. Samples will be analysed using 1H NMR spectroscopy and the resultant metabolomic profiles will be subject to multivariate pattern recognition techniques. This will produce mathematical models capable of distinguishing these long covid and control groups. Symptoms, potential confounders, and qualitative narrative data will be collected alongside this process to add deeper richness to the subsequent analysis. Primary Outcome The creation of a diagnostic test for long covid using 1H NMR metabolomics. Secondary Outcomes The development of algorithms that predict the severity and chronicity of long covid, identification of subgroup differences in metabolomic and immune profiles, and triangulation with symptom and narrative data to produce a deeper understanding of the patient experience. Conclusion This study seeks to advance the understanding of long covid using advanced multi-omic and narrative techniques, which may offer potential diagnostic and therapeutic avenues.
CaV2.1 mediates presynaptic dysfunction induced by amyloid β oligomers.
Synaptic dysfunction is an early pathological phenotype of Alzheimer's disease (AD) that is initiated by oligomers of amyloid β peptide (Aβos). Treatments aimed at correcting synaptic dysfunction could be beneficial in preventing disease progression, but mechanisms underlying Aβo-induced synaptic defects remain incompletely understood. Here, we uncover an epithelial sodium channel (ENaC) - CaV2.3 - protein kinase C (PKC) - glycogen synthase kinase-3β (GSK-3β) signal transduction pathway that is engaged by Aβos to enhance presynaptic CaV2.1 voltage-gated Ca2+ channel activity, resulting in pathological potentiation of action-potential-evoked synaptic vesicle exocytosis. We present evidence that the pathway is active in human APP transgenic mice in vivo and in human AD brains, and we show that either pharmacological CaV2.1 inhibition or genetic CaV2.1 haploinsufficiency is sufficient to restore normal neurotransmitter release. These findings reveal a previously unrecognized mechanism driving synaptic dysfunction in AD and identify multiple potentially tractable therapeutic targets.
Comprehensive analysis of SLC17A5 variants in large European cohorts reveals no association with Parkinson's disease risk.
BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss and α-synuclein aggregation. Aging is the primary risk factor, with both rare and common genetic variants playing a role. Previous studies have implicated lysosomal storage disorder (LSD)-related genes, including SLC17A5, in PD susceptibility. OBJECTIVE: This study aimed to investigate the association of SLC17A5 variants, including rare and common variants and the FSASD-associated p.Arg39Cys missense variant, with PD risk in large European ancestry cohorts. METHODS: Rare variant burden analyses were performed at minor allele frequency (MAF) thresholds of ≤1 % and ≤0.1 % in 7,184 PD cases and 51,650 controls using whole-genome and whole-exome sequencing data. Association testing of the p.Arg39Cys variant was conducted across five cohorts, encompassing both Finnish and non-Finnish Europeans. Common variant associations were examined using summary statistics from the largest European GWAS of PD. RESULTS: No significant association was observed between rare SLC17A5 variants and PD at either MAF threshold. The p.Arg39Cys variant, though enriched in Finnish Europeans, showed no significant association with PD across several cohorts. Similarly, common SLC17A5 variants (MAF ≥1%) were not associated with PD risk. CONCLUSION: Our findings do not support a role for SLC17A5 variants in PD susceptibility. While lysosomal dysfunction is central to PD pathogenesis, its contribution appears pathway-specific, with SLC17A5 unlikely to influence risk. Larger, multiethnic studies and functional analyses are needed to further investigate sialic acid metabolism in PD and related disorders.
Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic β-cells.
Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic β-cells. This underlies the progressive decline in β-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces β-cell function.
Circulating neuropeptide Y dynamics and performance during exercise in heart failure patients with contemporary medical and device therapy.
High cardiac sympathetic drive and release of the sympathetic cotransmitter neuropeptide Y (NPY) are significant features of congestive heart failure (CHF), in which resting venous NPY levels are known to be associated with mortality. However, whether circulating NPY levels increase during exercise in CHF when they are already elevated is controversial. We sought to establish the dynamics of circulating NPY levels in CHF patients treated with contemporary medical therapy and devices in relationship to indices of performance linked to long-term prognosis. CHF patients (n = 15) underwent cardiopulmonary exercise testing with venous blood sampling at rest, peak exercise and recovery. These patients had significantly higher resting venous NPY levels compared with an age- and sex-matched control group of patients (n = 16) with normal left ventricular function (40 ± 6.9 vs. 9.0 ± 4.6 pg/mL, respectively; P
Unique pathways downstream of TLR-4 and TLR-7 activation: sex-dependent behavioural, cytokine, and metabolic consequences.
INTRODUCTION: Post-infection syndromes are characterised by fatigue, muscle pain, anhedonia, and cognitive impairment; mechanistic studies exploring these syndromes have focussed on pathways downstream of Toll-like receptor (TLR) 4 activation. Here, we investigated the mechanistic interplay between behaviour, metabolism, and inflammation downstream of TLR-7 activation compared to TLR-4 activation in male and female CD1 mice. METHODS: Animals received either a TLR-4 (LPS; 0.83 mg/kg) or TLR-7 (R848, 5 mg/kg) agonist, or saline, and behaviour was analysed in an Open Field (OF) at 24 h (n = 20/group). Plasma, liver, and prefrontal cortex (PFC) were collected for gene expression analysis at 24 h and 1H-NMR metabolomics. RESULTS: TLR-4 and TLR-7 activation decreased distance travelled and rearing in the OF, but activation of each receptor induced distinct cytokine responses and metabolome profiles. LPS increased IL-1β expression and CXCL1 in the PFC, but TLR7 activation did not and strongly induced PFC CXCL10 expression. Thus, TLR7 induced sickness behaviour is independent of IL-1β expression. In both cases, the behavioural response to TLR activation was sexually dimorphic: females were more resilient. However, dissociation was observed between the resilient female mice behaviour and the levels of gene cytokine expression, which was, in general, higher in the female mice. However, the metabolic shifts induced by immune activation were better correlated with the sex-dependent behavioural dimorphisms; increased levels of antioxidant potential in the female brain are intrinsic male/female metabolome differences. A common feature of both TLR4 and TLR7 activation was an increase in N-acetyl aspartate (NAA) in the PFC, which is likely be an allostatic response to the challenges as sickness behaviour is inversely correlated with NAA levels. DISCUSSION: The results highlight how the cytokine profile induced by one PAMP cannot be extrapolated to another, but they do reveal how the manipulation of the conserved metabolome response might afford a more generic approach to the treatment of post-infection syndromes.
TPC2 in drug development: Emerging target for cancer, viral infections, cardiovascular diseases, and neurological disorders.
The lysosomal two-pore channel 2 (TPC2) modulates intracellular calcium (Ca2 +) signaling and has been implicated in inflammatory, cardiovascular, and neurodegenerative conditions, as well as cancer and viral infections. Despite its potential as a drug target, TPC2 is still in the early stages of therapeutic development. The major challenges include achieving high target specificity without inducing unintended effects on other endolysosomal channels and on the crosstalk between TPC2 and other intracellular and extracellular Ca2+ channels. Recent advancements in the structural analysis of TPC2, along with the development of TPC2 agonists and inhibitors, have significantly expanded our understanding of its mechanistic contributions to disease. This review highlights potential TPC2-based therapies for cancer, inflammation, and neurological disorders, emphasizing the need for further research to develop targeted TPC2 modulators and fully elucidate the molecular mechanisms of TPC2.