Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells.

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.

Gene therapy for Lafora disease in the Epm2a-/- mouse model.

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. The disease results from mutations in the EPM2A gene, encoding laforin, or the EPM2B gene, encoding malin. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein cause alterations in this complex, leading to the formation of Lafora bodies containing abnormal, insoluble, and hyperphosphorylated forms of glycogen. We used the Epm2a-/- knockout mouse model of Lafora disease to apply gene therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment through neuropathological studies, behavioral tests, video-electroencephalography, electrophysiological recordings, and proteomic/phosphoproteomic analysis. Gene therapy ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Moreover, differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Our results represent proof of principle for gene therapy with the coding region of the human EPM2A gene as a treatment for EPM2A-related Lafora disease.

Mutagenesis of Wheat Powdery Mildew Reveals a Single Gene Controlling Both NLR and Tandem Kinase-Mediated Immunity.

Blumeria graminis f. sp. tritici (Bgt) is a globally important fungal wheat pathogen. Some wheat genotypes contain powdery mildew resistance (Pm) genes encoding immune receptors that recognize specific fungal-secreted effector proteins, defined as avirulence (Avr) factors. Identifying Avr factors is vital for understanding the mechanisms, functioning, and durability of wheat resistance. Here, we present AvrXpose, an approach to identify Avr genes in Bgt by generating gain-of-virulence mutants on Pm genes. We first identified six Bgt mutants with gain of virulence on Pm3b and Pm3c. They all had point mutations, deletions or insertions of transposable elements within the corresponding AvrPm3b2/c2 gene or its promoter region. We further selected six mutants on Pm3a, aiming to identify the yet unknown AvrPm3a3 recognized by Pm3a, in addition to the previously described AvrPm3a2/f2. Surprisingly, Pm3a virulence in the obtained mutants was always accompanied by an additional gain of virulence on the unrelated tandem kinase resistance gene WTK4. No virulence toward 11 additional R genes tested was observed, indicating that the gain of virulence was specific for Pm3a and WTK4. Several independently obtained Pm3a-WTK4 mutants have mutations in Bgt-646, a gene encoding a putative, nonsecreted ankyrin repeat-containing protein. Gene expression analysis suggests that Bgt-646 regulates a subset of effector genes. We conclude that Bgt-646 is a common factor required for avirulence on both a specific nucleotide-binding leucine-rich repeat and a WTK immune receptor. Our findings suggest that, beyond effectors, another type of pathogen protein can control the race-specific interaction between powdery mildew and wheat. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Haploinsufficiency of the mouse Tshz3 gene leads to kidney defects.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Network analytics for drug repurposing in COVID-19.

To better understand the potential of drug repurposing in COVID-19, we analyzed control strategies over essential host factors for SARS-CoV-2 infection. We constructed comprehensive directed protein-protein interaction (PPI) networks integrating the top-ranked host factors, the drug target proteins and directed PPI data. We analyzed the networks to identify drug targets and combinations thereof that offer efficient control over the host factors. We validated our findings against clinical studies data and bioinformatics studies. Our method offers a new insight into the molecular details of the disease and into potentially new therapy targets for it. Our approach for drug repurposing is significant beyond COVID-19 and may be applied also to other diseases.

Tumoral periprostatic adipose tissue exovesicles-derived miR-20a-5p regulates prostate cancer cell proliferation and inflammation through the RORA gene.

BACKGROUND: From the first steps of prostate cancer (PCa) initiation, tumours are in contact with the most-proximal adipose tissue called periprostatic adipose tissue (PPAT). Extracellular vesicles are important carriers of non-coding RNA such as miRNAs that are crucial for cellular communication. The secretion of extracellular vesicles by PPAT may play a key role in the interactions between adipocytes and tumour. Analysing the PPAT exovesicles (EVs) derived-miRNA content can be of great relevance for understanding tumour progression and aggressiveness. METHODS: A total of 24 samples of human PPAT and 17 samples of perivesical adipose tissue (PVAT) were used. EVs were characterized by western blot and transmission electron microscopy (TEM), and uptake by PCa cells was verified by confocal microscopy. PPAT and PVAT explants were cultured overnight, EVs were isolated, and miRNA content expression profile was analysed. Pathway and functional enrichment analyses were performed seeking potential miRNA targets. In vitro functional studies were evaluated using PCa cells lines, miRNA inhibitors and target gene silencers. RESULTS: Western blot and TEM revealed the characteristics of EVs derived from PPAT (PPAT-EVs) samples. The EVs were up taken and found in the cytoplasm of PCa cells. Nine miRNAs were differentially expressed between PPAT and PVAT samples. The RORA gene (RAR Related Orphan Receptor A) was identified as a common target of 9 miRNA-regulated pathways. In vitro functional analysis revealed that the RORA gene was regulated by PPAT-EVs-derived miRNAs and was found to be implicated in cell proliferation and inflammation. CONCLUSION: Tumour periprostatic adipose tissue is linked to PCa tumour aggressiveness and could be envisaged for new therapeutic strategies.

A diverse panel of 755 bread wheat accessions harbors untapped genetic diversity in landraces and reveals novel genetic regions conferring powdery mildew resistance.

KEY MESSAGE: A bread wheat panel reveals rich genetic diversity in Turkish, Pakistani and Iranian landraces and novel resistance loci to diverse powdery mildew isolates via subsetting approaches in association studies. Wheat breeding for disease resistance relies on the availability and use of diverse genetic resources. More than 800,000 wheat accessions are globally conserved in gene banks, but they are mostly uncharacterized for the presence of resistance genes and their potential for agriculture. Based on the selective reduction of previously assembled collections for allele mining for disease resistance, we assembled a trait-customized panel of 755 geographically diverse bread wheat accessions with a focus on landraces, called the LandracePLUS panel. Population structure analysis of this panel based on the TaBW35K SNP array revealed an increased genetic diversity compared to 632 landraces genotyped in an earlier study and 17 high-quality sequenced wheat accessions. The additional genetic diversity found here mostly originated from Turkish, Iranian and Pakistani landraces. We characterized the LandracePLUS panel for resistance to ten diverse isolates of the fungal pathogen powdery mildew. Performing genome-wide association studies and dividing the panel further by a targeted subsetting approach for accessions of distinct geographical origin, we detected several known and already cloned genes, including the Pm2a gene. In addition, we identified 22 putatively novel powdery mildew resistance loci that represent useful sources for resistance breeding and for research on the mildew-wheat pathosystem. Our study shows the value of assembling trait-customized collections and utilizing a diverse range of pathogen races to detect novel loci. It further highlights the importance of integrating landraces of different geographical origins into future diversity studies.

Analysis of a global wheat panel reveals a highly diverse introgression landscape and provides evidence for inter-homoeologue chromosomal recombination.

KEY MESSAGE: This study highlights the agronomic potential of rare introgressions, as demonstrated by a major QTL for powdery mildew resistance on chromosome 7D. It further shows evidence for inter-homoeologue recombination in wheat. Agriculturally important genes are often introgressed into crops from closely related donor species or landraces. The gene pool of hexaploid bread wheat (Triticum aestivum) is known to contain numerous such "alien" introgressions. Recently established high-quality reference genome sequences allow prediction of the size, frequency and identity of introgressed chromosome regions. Here, we characterise chromosomal introgressions in bread wheat using exome capture data from the WHEALBI collection. We identified 24,981 putative introgression segments of at least 2 Mb across 434 wheat accessions. Detailed study of the most frequent introgressions identified T. timopheevii or its close relatives as a frequent donor species. Importantly, 118 introgressions of at least 10 Mb were exclusive to single wheat accessions, revealing that large populations need to be studied to assess the total diversity of the wheat pangenome. In one case, a 14 Mb introgression in chromosome 7D, exclusive to cultivar Pamukale, was shown by QTL mapping to harbour a recessive powdery mildew resistance gene. We identified multiple events where distal chromosomal segments of one subgenome were duplicated in the genome and replaced the homoeologous segment in another subgenome. We propose that these examples are the results of inter-homoeologue recombination. Our study produced an extensive catalogue of the wheat introgression landscape, providing a resource for wheat breeding. Of note, the finding that the wheat gene pool contains numerous rare, but potentially important introgressions and chromosomal rearrangements has implications for future breeding.

Autoimmune inflammation triggers aberrant astrocytic calcium signaling to impair synaptic plasticity.

Cortical pathology involving inflammatory and neurodegenerative mechanisms is a hallmark of multiple sclerosis and a correlate of disease progression and cognitive decline. Astrocytes play a pivotal role in multiple sclerosis initiation and progression but astrocyte-neuronal network alterations contributing to gray matter pathology remain undefined. Here we unveil deregulation of astrocytic calcium signaling and astrocyte-to-neuron communication as key pathophysiological mechanisms of cortical dysfunction in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Using two-photon imaging ex vivo and fiber photometry in freely behaving mice, we found that acute EAE was associated with the emergence of spontaneously hyperactive cortical astrocytes exhibiting dysfunctional responses to cannabinoid, glutamate and purinoreceptor agonists. Abnormal astrocyte signaling by Gi and Gq protein coupled receptors was observed in the inflamed cortex. This was mirrored by treatments with pro-inflammatory factors both in vitro and ex vivo, suggesting cell-autonomous effects of the cortical neuroinflammatory environment. Finally, deregulated astrocyte calcium activity was associated with an enhancement of glutamatergic gliotransmission and a shift of astrocyte-mediated short-term and long-term plasticity mechanisms towards synaptic potentiation. Overall, our data identify astrocyte-neuronal network dysfunctions as key pathological features of gray matter inflammation in multiple sclerosis and potentially additional neuroimmunological disorders.

Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia.

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

The role of supervision and motivation during exercise on physical and mental health in older adults: a study protocol for a randomized controlled trial (PRO-Training project).

BACKGROUND: Although supervised exercise is frequently recommended for older adults, its superiority over unsupervised exercise remains uncertain. Furthermore, whether motivational techniques could help to enhance the effectiveness of the latter remains to be elucidated. The present randomized controlled trial aims to determine the role of supervision and motivational strategies on the safety, adherence, efficacy, and cost-effectiveness of different exercise programs for improving physical and mental health in older adults. METHODS: Participants (n = 120, aged 60-75 years) will be randomly allocated into five groups: 1-Control (CON), 2-Supervised exercise without motivational intervention (SUP), 3- Supervised exercise with motivational intervention (SUP +), 4- Unsupervised exercise without motivational intervention (UNSUP) and 5- Unsupervised exercise with motivational intervention (UNSUP +). Over 24 weeks, all exercise groups will participate in a multicomponent exercise program three times/week (performed in group classes at a center for SUP and SUP + , or home without supervision but with the help of a mobile app for UNSUP and UNSUP +), while the CON group will maintain their usual lifestyle. The motivational intervention (for SUP + and UNSUP + groups) will be based on the self-determination theory, including strategies such as phone calls, interactive workshops, motivational messages, informative infographics and videos. Primary outcomes will include safety, adherence, costs, and lower-body muscular function using a leg press machine. Secondary outcomes will include upper-body muscular function, physical and cardiorespiratory function, blood pressure and heart rate, body composition, health-related quality of life, cognitive performance, anxiety, depression, physical activity levels, sleep and sedentarism, biochemical markers, motivators and barriers to exercise. Assessments will be conducted at baseline, mid-intervention (i.e., week 13), at the end of the intervention (i.e., week 25), and 24 weeks later (i.e., week 49). DISCUSSION: The findings of this trial might provide valuable insights into the role of supervision and motivational strategies on the effectiveness of exercise programs for older adults. Additionally, the study could contribute to developing cost-effective interventions, supporting the design of future public policies for healthy aging. TRIAL REGISTRATION: NCT05619250. Registered 16 November 2022.

Precision Medicine in Childhood Cancer: The Influence of Genetic Polymorphisms on Vincristine-Induced Peripheral Neuropathy.

Cancer is the leading cause of disease-related death among children. Vincristine (VCR), a key component of childhood cancer treatment protocols, is associated with the risk of peripheral neuropathy (PN), a condition that may be reversible upon drug discontinuation but can also leave lasting sequelae. Single nucleotide polymorphism (SNP) in genes involved in VCR pharmacokinetics and pharmacodynamics have been investigated in relation to an increased risk of PN. However, the results of these studies have been inconsistent. A retrospective cohort study was conducted to investigate the potential association of drug transporter genes from the ATP-binding cassette (ABC) family and the centrosomal protein 72 (CEP72) gene with the development of PN in 88 Caucasian children diagnosed with cancer and treated with VCR. Genotyping was performed using real-time PCR techniques for the following SNPs: ABCB1 rs1128503, ABCC1 rs246240, ABCC2 rs717620, and CEP72 rs924607. The results indicated that age at diagnosis (OR = 1.33; 95% CI = 1.07-1.75) and the ABCC1 rs246240 G allele (OR = 12.48; 95% CI = 2.26-100.42) were associated with vincristine-induced peripheral neuropathy (VIPN). No association was found between this toxicity and CEP72 rs924607. Our study provides insights that may contribute to optimizing childhood cancer therapy in the future by predicting the risk of VIPN.

European survey on Paediatric Early Warning Systems, and other processes used to aid the recognition and response to children's deterioration on hospital wards.

BACKGROUND: Internationally, there is an increasing trend in using Rapid Response Systems (RRS) to stabilize in-patient deterioration. Despite a growing evidence base, there remains limited understanding of the processes in place to aid the early recognition and response to deteriorating children in hospitals across Europe. AIM/S: To describe the processes in place for early recognition and response to in-patient deterioration in children in European hospitals. STUDY DESIGN: A cross-sectional opportunistic multi-centre European study, of hospitals with paediatric in-patients, using a descriptive self-reported, web-based survey, was conducted between September 2021 and March 2022. The sampling method used chain referral through members of European and national societies, led by country leads. The survey instrument was an adaptation to the survey of Recognition and Response Systems in Australia. The study received ethics approval. Descriptive analysis and Chi-squared tests were performed to compare results in European regions. RESULTS: A total of 185 questionnaires from 21 European countries were received. The majority of respondents (n = 153, 83%) reported having written policies, protocols, or guidelines, regarding the measurement of physiological observations. Over half (n = 120, 65%) reported that their hospital uses a Paediatric Early Warning System (PEWS) and 75 (41%) reported having a Rapid Response Team (RRT). Approximately one-third (38%) reported that their hospital collects specific data about the effectiveness of their RRS, while 100 (54%) reported providing regular training and education to support it. European regional differences existed in PEWS utilization (North = 98%, Centre = 25%, South = 44%, p < .001) and process evaluation (North = 49%, Centre = 6%, South = 36%, p < .001). CONCLUSIONS: RRS practices in European hospitals are heterogeneous. Differences in the uptake of PEWS and RRS process evaluation emerged across Europe. RELEVANCE TO CLINICAL PRACTICE: It is important to scope practices for the safe monitoring and management of deteriorating children in hospital across Europe. To reduce variance in practice, a consensus statement endorsed by paediatric and intensive care societies could provide guidance and resources to support PEWS implementation and for the operational governance required for continuous quality improvement.

METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.

Epm2aR240X knock-in mice present earlier cognitive decline and more epileptic activity than Epm2a-/- mice.

Lafora disease is a rare recessive form of progressive myoclonic epilepsy, usually diagnosed during adolescence. Patients present with myoclonus, neurological deterioration, and generalized tonic-clonic, myoclonic, or absence seizures. Symptoms worsen until death, usually within the first ten years of clinical onset. The primary histopathological hallmark is the formation of aberrant polyglucosan aggregates called Lafora bodies in the brain and other tissues. Lafora disease is caused by mutations in either the EPM2A gene, encoding laforin, or the EPM2B gene, coding for malin. The most frequent EPM2A mutation is R241X, which is also the most prevalent in Spain. The Epm2a-/- and Epm2b-/- mouse models of Lafora disease show neuropathological and behavioral abnormalities similar to those seen in patients, although with a milder phenotype. To obtain a more accurate animal model, we generated the Epm2aR240X knock-in mouse line with the R240X mutation in the Epm2a gene, using genetic engineering based on CRISPR-Cas9 technology. Epm2aR240X mice exhibit most of the alterations reported in patients, including the presence of LBs, neurodegeneration, neuroinflammation, interictal spikes, neuronal hyperexcitability, and cognitive decline, despite the absence of motor impairments. The Epm2aR240X knock-in mouse displays some symptoms that are more severe that those observed in the Epm2a-/- knock-out, including earlier and more pronounced memory loss, increased levels of neuroinflammation, more interictal spikes and increased neuronal hyperexcitability, symptoms that more precisely resemble those observed in patients. This new mouse model can therefore be specifically used to evaluate how new therapies affects these features with greater precision.

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling.

In this Perspective, we discuss the current status and advances in spatial transcriptomics technologies, which allow high-resolution mapping of gene expression in intact cell and tissue samples. Spatial transcriptomics enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to the bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, and neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution utilizing diverse technological approaches. In this Perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest, and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques as well as provide information on data analysis. In addition, this Perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.