Instituto de Biomedicina de Valencia

Defects in chromosome synapsis and meiotic recombination activate a checkpoint that, in budding yeast, delays exit from meiotic prophase I by inhibiting the Ndt80-dependent expression of regulators including the cyclin CLB1 and the polo-like kinase (PLK) CDC5. Additionally, Swe1-mediated inhibitory phosphorylation of cyclin-dependent kinase 1 (Cdk1/Cdc28) reinforces this arrest. Once the checkpoint is released, Cdk1 activation is essential for meiosis I entry and requires removal of inhibitory phosphorylation on tyrosine 19, governed by the opposing activities of the Swe1 kinase and the Mih1 phosphatase. Here, we dissect how this network is rewired at the prophase I to meiosis I transition. We show that Swe1 is essential for checkpoint maintenance, but not for its initial activation. We also demonstrate that Cdc5 promotes Cdk1 activation through a dual mechanism: by inducing Swe1 degradation and facilitating Mih1 nuclear translocation. Unlike in mitosis, Cdc5-dependent Swe1 degradation in meiosis does not require CDK-mediated priming and can occur when both proteins are artificially colocalized, indicating a distinct regulatory mode. Our findings uncover a novel function for Cdc5 in promoting meiotic cell cycle progression beyond its known roles in recombination and synaptonemal complex disassembly, highlighting how conserved cell cycle regulators are adapted to meiosis.

Lafora disease is a fatal neurodegenerative disorder caused by loss-of-function mutations in the EPM2A or EPM2B genes, which encode laforin and malin, respectively. These mutations lead to the accumulation of intracellular inclusions of abnormal glycogen, known as Lafora bodies, the hallmark of the disease. Symptoms typically begin in early adolescence with seizures and rapidly progress to cognitive and motor decline, ultimately resulting in dementia and death within a decade of onset. Disruption of Epm2a or Epm2b in mice causes neuronal degeneration and Lafora body accumulation in the brain and other tissues. Epm2a−/− and Epm2b−/− mice exhibit motor and memory impairments, epileptic activity, and molecular and histological abnormalities. We previously demonstrated that intracerebroventricular delivery of a recombinant adeno-associated virus carrying EPM2A significantly improved pathology in Epm2a−/− mice. In this study, we tested recombinant adeno-associated virus-mediated delivery of the human EPM2B gene in Epm2b−/− mice. The treatment partially improved neurological, molecular, and histopathological outcomes, although some pathological features persisted. Importantly, our findings reveal differences between EPM2A- and EPM2B-based gene therapies, highlighting the need to better understand their distinct mechanisms. Despite limitations, our study provides new insights into the complexity of targeting EPM2B mutations in Lafora disease.

Abstract Antimicrobial resistance (AMR) represents a critical threat to global health, largely driven by the dissemination of β-lactamases that inactivate frontline antibiotics. Among the most problematic are BlaMab-2 from Mycobacterium abscessus , KPC-2 and OXA-48 from Klebsiella pneumoniae , and VIM-2 from Pseudomonas aeruginosa , which together confer broad resistance to β-lactams and carbapenems. Current β-lactamase inhibitors face declining efficacy as resistance variants continue to emerge, underscoring the need for innovative strategies. Here, we explored single-domain antibodies (sd-Abs) as enzyme-directed inhibitors of β-lactamases. A library of sd-Abs was screened, and two candidates, B2 and B5, were characterized in vitro and in vivo . Both sd-Abs inhibited BlaMab-2 activity in E. coli expression systems, following a competitive inhibition mechanism, with B2 consistently displaying stronger potency (K i ≈ 1.5 µM) than B5. Remarkably, B2 also demonstrated broad inhibitory activity against KPC-2, VIM-2, and OXA-48, while B5 showed an alternative inhibition profile, including uncompetitive characteristics against VIM-2 and OXA-48. Comparison with clinically deployed inhibitors revealed that the K i values of B2 and B5 are of the same order of magnitude—or superior in some cases—highlighting their therapeutic promise. Our findings establish sd-Abs as a versatile platform for the inhibition of diverse β-lactamases, with B2 emerging as the most broadly effective candidate. By expanding the utility of existing β-lactams, sd-Abs could help restore antibiotic efficacy against multidrug-resistant pathogens. This study underscores the potential of antibody-based enzyme inhibitors as a new class of anti-resistance therapeutics.

This study presents an improved loop-mediated isothermal amplification (LAMP) procedure for the quick, inexpensive, and precise identification and quantification of lactic acid bacteria (LAB) in white and red wines and in grape musts. Custom primers targeting the 16S rRNA gene were developed to enhance specificity. Key advancements include a mobile phone-based semi-quantification approach using hydroxy naphthol blue (HNB), endpoint quantification using a microplate reader, and a real-time quantitative LAMP (qLAMP) assay using EvaGreen®. Moreover, PMA-qLAMP was optimised to differentiate viable from non-viable bacterial cells. A major advantage arises from the fact that all red and white wine and must samples examined exhibited consistent behaviours. The LAMP improvements described provide wineries with accessible tools for microbial quality control by themselves, offering a practical alternative to traditional methods requiring sophisticated instrumentation.

ObjectiveCharcot–Marie–Tooth (CMT) disease is a heterogeneous group of genetic neuropathies, with >90 genes identified. Several aminoacyl‐tRNA synthetases have been linked to CMT. DARS2, encoding the mitochondrial aspartyl‐tRNA synthetase, has been typically associated with leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation. This study aimed to investigate the association between biallelic DARS2 variants and axonal CMT.MethodsWe investigated 5 individuals from 3 unrelated families with axonal CMT and biallelic DARS2 variants. Functional studies in fibroblasts assessed their effects on DARS2 expression, localization, and mitochondrial function. Enzymatic activity was evaluated in HEK293 cells.ResultsThe 5 individuals, including 4 adults, presented with childhood‐onset progressive axonal CMT. None had leukoencephalopathy, but one showed central nervous system involvement, with intellectual disability and epilepsy. Genetic analysis identified compound heterozygous DARS2 variants: family A, p.Ser238Phe and p.Arg336Cys; family B, p.Ser238Phe and p.Ile25Thrfs*38; family C, c.492+2T>C and p.Pro503Leu. Functional studies revealed reduced DARS2 protein levels, mitochondrial network abnormalities, and impaired mitochondrial function. p.Ser238Phe behaves as a hypomorphic allele, whereas p.Pro503Leu reduced DARS2 enzymatic activity by 75%.InterpretationOur findings expand the DARS2‐related disease spectrum, establishing a novel association with axonal CMT. Hypomorphic variants, such as p.Ser238Phe, when paired with more deleterious variants, result in isolated axonal CMT, whereas more severe combinations—although not as deleterious as those seen in leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation—result in axonal CMT with central nervous system involvement, albeit without leukoencephalopathy. These observations raise the possibility that DARS2‐associated diseases form a continuum rather than representing strictly distinct central or peripheral nervous system disorders. ANN NEUROL 2025;98:1335–1351

Originally known for its function in the cell cycle, the anaphase-promoting complex/cyclosome (APC/C) also plays a crucial role in regulating differentiation and maintaining cell identity. However, the mechanisms by which APC/C mediates developmental processes are not fully understood. In this study, we show that APC/C and its activator FZR-1 regulate the chromatin regulators MES-4 and MES-3. These proteins are part of histone methylation complexes essential for maintaining germline stem cell (GSC) identity in the germ line of Caenorhabditis elegans. APC/CFZR-1 facilitates the degradation of MES-4 and MES-3 when GSCs transition toward differentiating into oocytes. The activity of APC/CFZR-1 is restricted by the Notch signaling pathway provided by the distal tip cell, which is responsible for maintaining the stemness of the GSC pool. This negative regulation enables the accumulation of MES-3 and MES-4 in GSCs, offering an additional component by which niche activity modulates the C. elegans germ line.

Abstract Insect mass-rearing is a rapidly expanding industry for the production of protein for food and feed. These highly productive artificial rearing environments can expose insects to a range of biotic and abiotic stressors, including insect pathogens, which may result in population crashes. Interactions between insect pathogens with multiple stressors can exacerbate the effects of individual pathogens on host insects. However, reliable predictions on the combined effects of individual stressors based on mechanisms of action are lacking within the field of insect pathology. We review how ecotoxicological modelling of multiple stressors can be applied to mass-reared insect systems and discuss the importance of standardization across research fields investigating multiple stressors. Important considerations in multiple stressor terminology, experimental design, endpoints and analysis of results are discussed to improve understanding of multiple stressors and their impact on insects for food and feed. This is essential for ensuring optimal rearing conditions for mass-reared insect populations.

Urea cycle disorders (UCDs) are inherited diseases causing recurrent life-threatening metabolic decompensations due to impaired hepatic ammonia detoxification and decreased ureagenesis. Ornithine transcarbamylase (OTC) deficiency (OTCD) is X-linked and the most common and often fatal UCD. In male hemizygous patients, disease severity primarily depends on the pathogenic sequence variant, while in heterozygous females, disease severity also depends on the X-chromosomal inactivation (XCI) pattern. Females with unfavorable XCI predominantly expressing the mutant OTC protein may be severely affected. Here, we investigated a novel treatment strategy for OTCD since there is an unmet need for better therapies. In the first step, we performed a high throughput screening (HTS) using a diversity library with 10 000 chemical compounds to identify pharmacological chaperone (PC) candidates that stabilize purified wild-type OTC. Stratification of our HTS results revealed five potential PCs, which were selected for further experimentation in cellular systems using primary human hepatocytes (PHHs) and human induced pluripotent stem cell (hiPSC)-derived hepatocytes (hiPSC-Heps) from healthy controls and OTCD patients. Two PCs-PC1 and PC4-increased OTC protein stability and activity in control hiPSC-Heps, while PC4 in addition increased OTC activity in patient-derived PHHs from a female OTCD patient with unfavorable XCI. Finally, PC1 and PC4 both significantly increased ureagenesis in patient-derived PHHs. To conclude, we identified two PCs that stabilized wild-type OTC and enhanced enzyme activity and ureagenesis. Our work suggests that PCs could provide a novel treatment strategy for OTCD specifically in females with unfavorable XCI.