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Objectives: We sought to assess mortality and morbidity in pregnant women with hypertrophic cardiomyopathy (HCM).Background: The risk associated with pregnancy in women with HCM is an important and increasingly frequent clinical issue for which systematic data are not available and a large measure of uncertainty persists.Methods: Maternal mortality in 91 consecutively evaluated families with HCM was compared with that reported in the general population. The study cohort included 100 women with HCM with one or more live births, for a total of 199 live births. Morbidity related to HCM during pregnancy was investigated in 40 women evaluated within five years of their pregnancy.Results: Two pregnancy-related deaths occurred, both in patients at a particularly high risk. The maternal mortality rate was 10 per 1,000 live births (95% confidence interval [CI] 1.1 to 36.2/1,000) and was in excess of the expected mortality in the general Italian population (relative risk 17.1, 95% CI 2.0 to 61.8). In the 40 patients evaluated within close proximity of their pregnancy, 1 (4%) of the 28 who were previously asymptomatic and 5 (42%) of the 12 with symptoms progressed to functional class III or IV during pregnancy (p < 0.01). One patient had atrial fibrillation and one had syncope, both of whom had already experienced similar and recurrent events before their pregnancy.Conclusions: Maternal mortality is increased in patients with HCM compared with the general population. However, absolute maternal mortality is low and appears to be principally confined to women at a particularly high risk. In the presence of a favorable clinical profile, the progression of symptoms, atrial fibrillation, and syncope are also uncommon during pregnancy. [ABSTRACT FROM AUTHOR]

Patients presenting with ischemic chest pain and electrocardiographic evidence of global T-wave inversion are most frequently women with intact left ventricular function and no critical stenosis of major coronary vessels. Hence, this syndrome has a good immediate and long-term prognosis. [ABSTRACT FROM AUTHOR]

LARP4 interacts with poly(A)-binding protein (PABP) to protect messenger RNAs (mRNAs) from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved and relevance had been undetermined. Here, through a combination of in-cell and in vitro methodologies, we identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and 646-656 in LARP4B) as directly binding RACK1. Consistent with these results, AlphaFold2-Multimer predicted high-confidence interaction of CR2 with RACK1 propellers 5 and 6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas PABP association was less affected, consistent with independent interactions. The CR2 mutations decreased LARP4's ability to stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) to higher degree than β-globin and GFP (green fluorescent protein) mRNAs lacking the ARE. We show LARP4 robustly increases translation of β-glo-ARE mRNA, whereas the LARP4 CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency, while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs., (Published by Oxford University Press on behalf of Nucleic Acids Research 2025.)

LARP4 interacts with poly(A)-binding protein (PABP) to protect mRNAs from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved, and relevance had been undetermined. Here, yeast two-hybrid domain mapping followed by other methods identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and LARP4B) as directly binding RACK1 region 200-317. Consistent with these results, AlphaFold2-multimer predicted high confidence interaction of CR2 with RACK1 propellers 5-6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas less effect was observed for PABP association, consistent with independent interactions. CR2 mutations decreased LARP4 ability to optimally stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) more significantly than a β-globin and other reporters lacking this element. While polysome profiles indicate the β-glo-ARE mRNA is inefficiently translated, consistent with published data, we show that LARP4 increases its translation whereas the LARP4-CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs., Competing Interests: COMPETING INTERESTS. The authors declare no competing interests.

RNA-binding proteins (RBPs) are emerging as important regulators of cancer pathogenesis. We reveal that the RBPs LARP4A and LARP4B are differentially overexpressed in osteosarcoma and osteosarcoma lung metastases, as well as in prostate cancer. Depletion of LARP4A and LARP4B reduced tumor growth and metastatic spread in xenografts, as well as inhibiting cell proliferation, motility, and migration. Transcriptomic profiling and high-content multiparametric analyses unveiled a central role for LARP4B, but not LARP4A, in regulating cell cycle progression in osteosarcoma and prostate cancer cells, potentially through modulating key cell cycle proteins such as Cyclins B1 and E2, Aurora B, and E2F1. This first systematic comparison between LARP4A and LARP4B assigns new pro-tumorigenic functions to LARP4A and LARP4B in bone and prostate cancer, highlighting their similarities while also indicating distinct functional differences. Uncovering clear biological roles for these paralogous proteins provides new avenues for identifying tissue-specific targets and potential druggable intervention., Competing Interests: The authors declare that they have no competing interests., (© 2024.)

Intermittent (or bolus) feeding regimens in critically ill patients have been of increasing interest to clinicians and scientists. Changes in amino acid, fat and carbohydrate metabolites over time might yet deliver other benefits (e.g. modulation of the circadian rhythm and sleep, and impacts on ghrelin secretion, insulin resistance and autophagy). We set out to characterise these changes in metabolite concentration. The Intermittent versus Continuous Feeding in Critically Ill paitents study (NCT02358512) was an eight-centre single-blinded randomised controlled trial. Patients were randomised to received a continuous (control arm) or intermittent (6x/day, intervention arm) enteral feeding regimen. Blood samples were taken on trial days 1, 7 and 10 immediately before and 30 min after intermittent feeds, and at equivalent timepoints in the control arm. A pre-planned targeted metabolomic analysis was performend using Nuclear Resonance Spectroscopy. Five hundred and ninety four samples were analysed from 75 patients. A total of 24 amino acid-, 19 lipid based-, and 44 small molecule metabolite features. Across the main two axes of variation (40-60% and 6-8% of variance), no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission. Logfold decreases in abundance were seen in metabolites related to amino acids (Glutamine - 0.682; Alanine - 0.594), ketone body metabolism (Acetone - 0.64; 3-Hydroxybutyric Acid - 0.632; Acetonacetic Acid - 0.586), fatty acid (carnitine - 0.509) and carbohydrate metabolism ( Maltose - 0.510; Citric Acid - 0.485). 2-3 Butanediol, a by-product of sugar-fermenting microbial metabolism also decreased (- 0.489). No correlation was seen with change in quadriceps muscle mass for any of the 20 metabolites varying with time (all p > 0.05). Increasing severity of organ failure was related to increasing ketone body metabolism (3 Hydroxybutyric Acid-1 and - 3; p = 0.056 and p = 0.014), carnitine deficiency (p = 0.002) and alanine abundancy (p - 0.005). A 6-times a day intermittent feeding regimen did not alter metabolite patterns across time compared to continuous feeding in critically ill patients, either within a 24 h period or across 10 days of intervention. Future research on intermittent feeding regimens should focus on clinical process benefits, or extended gut rest and fasting., (© 2023. The Author(s).)

Recent developments have mounted a stunning body of evidence underlying the importance of RNA binding proteins (RBPs) in cancer research. In this minireview we focus on LARP4A and LARP4B, two paralogs belonging to the superfamily of La-related proteins, and provide a critical overview of current research, including their roles in cancer pathogenesis and cell proliferation, migration, cell cycle and apoptosis. We highlight current controversies surrounding LARP4A and LARP4B and conclude that their complex roles in tumorigenesis are cell-, tissue- and context-dependent, warning that caution must be exercised before categorising either protein as an oncoprotein or tumour-suppressor. We also reveal that LARP4A and LARP4B have often been confused with one another, adding uncertainty in delineating their functions. We suggest that further functional and mechanistic studies of LARP4 proteins present significant challenges for future investigations to recognise the vital contributions of these RBPs in cancer research., Competing Interests: Declaration of Competing Interest The authors declare there are no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton., (© 2023. The Author(s).)

Medulloblastoma (MB) is the most common malignant brain tumour in children. High-risk MB patients harbouring MYC amplification or overexpression exhibit a very poor prognosis. Aberrant activation of MYC markedly reprograms cell metabolism to sustain tumorigenesis, yet how metabolism is dysregulated in MYC-driven MB is not well understood. Growing evidence unveiled the potential of BET-bromodomain inhibitors (BETis) as next generation agents for treating MYC-driven MB, but whether and how BETis may affect tumour cell metabolism to exert their anticancer activities remains unknown. In this study, we explore the metabolic features characterising MYC-driven MB and examine how these are altered by BET-bromodomain inhibition. To this end, we employed an NMR-based metabolomics approach applied to the MYC-driven MB D283 and D458 cell lines before and after the treatment with the BETi OTX-015. We found that OTX-015 triggers a metabolic shift in both cell lines resulting in increased levels of myo-inositol, glycerophosphocholine, UDP-N-acetylglucosamine, glycine, serine, pantothenate and phosphocholine. Moreover, we show that OTX-015 alters ascorbate and aldarate metabolism, inositol phosphate metabolism, phosphatidylinositol signalling system, glycerophospholipid metabolism, ether lipid metabolism, aminoacyl-tRNA biosynthesis, and glycine, serine and threonine metabolism pathways in both cell lines. These insights provide a metabolic characterisation of MYC-driven childhood MB cell lines, which could pave the way for the discovery of novel druggable pathways. Importantly, these findings will also contribute to understand the downstream effects of BETis on MYC-driven MB, potentially aiding the development of new therapeutic strategies to combat medulloblastoma., (© 2023. The Author(s).)

The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ 12,14 -Prostaglandin-J 2 , via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity., Competing Interests: Declaration of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified 'β-clasp' structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Soluble epoxide hydrolase (sEH), an enzyme that broadly regulates the cardiovascular system, hydrolyses epoxyeicosatrienoic acids (EETs) to their corresponding dihydroxyeicosatrienoic acids (DHETs). We previously showed that endogenous lipid electrophiles adduct within the catalytic domain, inhibiting sEH to lower blood pressure in angiotensin II-induced hypertensive mice. As angiotensin II increases vascular H 2 O 2 , we explored sEH redox regulation by this oxidant and how this integrates with inhibition by lipid electrophiles to regulate vasotone. Kinetics analyses revealed that H 2 O 2 not only increased the specific activity of sEH but increased its affinity for substrate and increased its catalytic efficiency. This oxidative activation was mediated by formation of an intra-disulfide bond between C262 and C264, as determined by mass spectrometry and substantiated by biotin-phenylarsinate and thioredoxin-trapping mutant assays. C262S/264S sEH mutants were resistant to peroxide-induced activation, corroborating the disulfide-activation mechanism. The physiological impact of sEH redox state was determined in isolated arteries and the effect of the pro-oxidant vasopressor angiotensin II on arterial sEH redox state and vasodilatory EETs indexed in mice. Angiotensin II induced the activating intra-disulfide in sEH, causing a decrease in plasma EET/DHET ratios that is consistent with the pressor response to this hormone. Although sEH C262-C264 disulfide formation enhances hydrolysis of vasodilatory EETs, this modification also sensitized sEH to inhibition by lipid electrophiles. This explains why angiotensin II decreases EETs and increases blood pressure, but when lipid electrophiles are also present, that EETs are increased and blood pressure lowered., (Copyright © 2021 Queen Mary University of London. Published by Elsevier B.V. All rights reserved.)

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The La-related proteins (LaRPs) are a superfamily of eukaryotic RNA-binding proteins with important and varied roles. To understand LaRP functions it is essential to unravel the divergent features responsible for their RNA target selectivity, which underlie their distinct identities and cellular roles. LaRPs are built on a common structural module called the 'La-module' that acts as a main locus for RNA recognition. The La-module is comprised of two tethered domains whose relative structural and dynamic interplay has been proposed to regulate RNA-target selection, albeit the mechanistic underpinning of this recognition remains to be elucidated. A main unsolved conundrum is how conserved La-modules across LaRPs are able to bind to extremely diverse RNA ligands.In this work, we employed Small Angle X-ray Scattering (SAXS) to investigate several human LaRP La-modules in the absence and, where applicable, in the presence of their RNA target, with the aim to explore the structural dynamics of their RNA recognition and provide information on the architectural landscape accessible to these proteins. Integration of these SAXS experiments with prior X-ray crystallography and NMR data suggests that RNA binding is generally accompanied by a compaction and loss of flexibility of the La-module. Nonetheless, the La-modules appear to experience a considerably different degree of inherent flexibility in their apo state. Furthermore, although they all exist in discrete subsets of accessible populations in equilibrium, these vary from LaRP to LaRP and can be either extended or compact. We propose that these divergent features may be critical for RNA substrate discrimination.

The La-related proteins (LaRPs) are an ancient superfamily of RNA-binding proteins orchestrating the major fates of RNA, from processing and maturation to regulation of mRNA translation. LaRPs are instrumental in modulating complex assemblies where the RNA is bound, folded, processed, escorted and presented to the functional effectors often through recruitment of protein partners. This intricate web of protein-RNA and protein-protein interactions is enabled by the modular nature of the LaRPs, comprising several structured domains connected by flexible linkers, and other sequences lacking recognizable folded motifs. Recent structures, together with biochemical and biophysical studies, have provided insights into how each LaRP family has evolved unique mechanisms of RNA recognition, not only through the conserved RNA-binding unit, the La-module, but also mediated by other family-specific motifs. Furthermore, in a series of unexpected twists and turns, they have revealed that the dynamic and conformational interplay of multi-structured domains and disordered regions operate in unison to achieve RNA substrate discrimination. This review proposes a perspective of our current knowledge of the structure-function relationship of the LaRP superfamily.

Nowadays, it is possible to combine X-ray crystallography and fragment screening in a medium throughput fashion to chemically probe the surfaces used by proteins to interact and use the outcome of the screens to systematically design protein-protein inhibitors. To prove it, we first performed a bioinformatics analysis of the Protein Data Bank protein complexes, which revealed over 400 cases where the crystal lattice of the target in the free form is such that large portions of the interacting surfaces are free from lattice contacts and therefore accessible to fragments during soaks. Among the tractable complexes identified, we then performed single fragment crystal screens on two particular interesting cases: the Il1β-ILR and p38α-TAB1 complexes. The result of the screens showed that fragments tend to bind in clusters, highlighting the small-molecule hotspots on the surface of the target protein. In most of the cases, the hotspots overlapped with the binding sites of the interacting proteins.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.