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Background: An increased incidence of Pseudomonas aeruginosa in microbiological surveillance (MS) cultures from gastrointestinal endoscopes was detected between March 2020 and March 2023 in Tan Tock Seng Hospital Singapore., Aim: To describe the use of whole-genome sequencing (WGS) in this investigation., Methods: WGS was performed for all P. aeruginosa isolates with pairwise comparison of isolates to assess for genomic linkage. Comprehensive review of reprocessing practices and environmental sampling was performed., Findings: Twenty-two P. aeruginosa isolates were detected from endoscopic MS cultures. Fifteen (68%) isolates were available for WGS. Eighteen pairwise comparisons of isolates were made, of which 10 were found to be genomically linked. One endoscope had P. aeruginosa repeatedly cultured from subsequent MS that were genomically linked and persistent despite repeat endoscopic reprocessing, establishing the persistence of biofilm that could not be eradicated with routine reprocessing. All P. aeruginosa isolates cultured from other different endoscopes were genetically distinct. Investigation into reprocessing practices revealed the use of air/water valves connected to endoscopes during clinical use. Inspection of these valves revealed the presences of cracks and tears. All other environmental samples were negative., Conclusion: The WGS findings helped to deprioritize common source contamination and supported the hypothesis of biofilm build-up within endoscopes, leading to repeatedly positive MS cultures that were genomically linked. This was possibly related to incomplete reprocessing of the damaged air/water valves, resulting in biofilm build-up. All faulty valves were changed and subsequently cleaned separately with ultrasonic cleaning followed by sterilization which resolved this incident., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Palmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. Although the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets., Objectives: We sought to identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis., Methods: We performed a genome-wide association meta-analysis of 3 North-European cohorts (n = 1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP., Results: We found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P < 5 × 10 -6 ) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and T H 2-mediated diseases such as atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP., Conclusions: The first genome-wide association study of PPP points to a pathogenic role for deregulated T H 2 responses and cigarette smoking., Competing Interests: Disclosure statement This work was supported by NIHR BioResource Centre Maudsley, National Institute for Health Research Maudsley Biomedical Research Centre (BRC) at South London and Maudsley NHS Foundation Trust and Institute of Psychiatry, Psychology and Neuroscience (IoPPN), King’s College London. We gratefully acknowledge capital equipment funding from the Maudsley Charity (grant no. 980) and Guy’s and St Thomas’s Charity (grant no. STR130505). The work was also supported by the NIHR Manchester Biomedical Research Centre (grant no. NIHR203308). The Trøndelag Health Study (HUNT) is a collaboration between HUNT Research Centre (Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology), Trøndelag County Council, Central Norway Regional Health Authority, and the Norwegian Institute of Public Health. The genotyping in HUNT was financed by the National Institutes of Health; University of Michigan; the Research Council of Norway; the Liaison Committee for Education, Research and Innovation in Central Norway; and the Joint Research Committee between St Olavs Hospital and the Faculty of Medicine and Health Sciences, NTNU. The APRICOT trial was funded by the Efficacy and Mechanism Evaluation (EME) Programme, an MRC and NIHR partnership (grant no. EME 13/50/17). This study was supported by Boehringer-Ingelheim and by the Psoriasis Association (grant nos. BSTOP50/5 and PhD studentship ST3/20 to A.H.C.). Z.Q.L. was supported by the Talent Development Fund of KK Women’s and Children’s Hospital, Singapore. N.J.R. is an NIHR Senior Investigator and is also supported by NIHR Newcastle Biomedical Research Centre, NIHR Newcastle In Vitro Diagnostics Co-operative, and NIHR Newcastle Patient Safety Research Collaboration. The views expressed are those of the author(s) and not necessarily those of the NHS, Department of Health, or King’s College London. None of the funders was involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Disclosure of potential conflict of interest: F. Capon has received grants and consultancy fees from Boehringer-Ingelheim. A. D. Burden has received consultancy fees from Boehringer-Ingelheim. C. E. M. Griffiths has received research grants and/or honoraria from AbbVie, Almirall, Anaptysbio, Boehringer-Ingelheim, Bristol Meyers Squibb, Evelo, GlaxoSmithKline, Inmagene, Janssen, Lilly, ONO Pharmaceuticals, Novartis, Pfizer, and UCB. P. Baum and S. Visvanathan are Boehringer-Ingelheim employees. S. Wahie has had nonfinancial support (sponsorship to attend dermatology conferences) from Janssen, AbbVie, Novartis, Almirall, and UCB. J. N. Barker declares paid activities as an advisor and speaker for AbbVie, Amgen, Boehringer-Ingelheim, Bristol Myers Squibb, Johnson & Johnson, Lilly, and Novartis. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The spread of Carbapenemase-producing Organisms (CPO) remains a major threat globally. Within clinical settings, the existing method of determining gene load involves traditional culture to determine bacterial load and polymerase-chain-reaction-based Xpert Carba-R Assay to determine carbapenemase gene type. However, there is a need for a fast and accurate method of quantifying CPO colonisation to study the risk of persistent CPO carriage., Objective: This study evaluated the accuracy of Xpert Carba-R Ct value in estimating carbapenamase producing bacterial loads in stool samples., Methods: Stool samples were obtained from an ongoing study investigating the household transmission of CPO in Singapore. Stool samples lacking carbapenemase producing organisms were spiked with organism carrying a single carbapenemase gene (blaKPC, blaNDM, blaVIM, blaOXA-48(-like) or blaIMP-1) and serially diluted before being subjected to Xpert Carba-R assay and traditional culture. Standard curves with regression lines showing correlation between Ct values and plate counts were generated. The standard curves were validated with stool samples collected from patients., Results: The limit of detection of blaNDM, blaKPC, and blaOXA-48 was approximately 103 cfu/mL, while that of blaIMP-1 and blaVIM was approximately 104 cfu/mL. Validation of the blaNDM and blaOXA-48 curves revealed average delta values of 0.56 log(cfu/mL) (95% CI 0.24-0.88) and 0.80 log(cfu/mL) (95% CI 0.53-1.07), respectively., Conclusions: Our validation data for stool positive for blaNDM and blaOXA-48-type suggests that bacterial loads can be estimated within a reasonable range of error., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe 2+ and peroxidated lipids. Notably, the Fe 2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Background: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive., Methods: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection., Results: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection., Conclusions: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins., (© 2024. The Author(s).)

Pseudomonas aeruginosa ST308 clone has been reported to carry carbapenemase genes such as bla IMP and bla VIM but has been rarely associated with bla NDM-1 . A total of 199 P. aeruginosa ST308 clinical and environmental isolates obtained between April 2019 and November 2020 from a tertiary-care hospital in Singapore were characterized using whole-genome sequencing. In addition, 71 bla NDM-1 -positive ST308 whole-genome sequences from two other local tertiary-care hospitals in Singapore and 83 global bla NDM-1 -negative ST308 whole-genome sequences in public databases were included to assess phylogenetic relationships and perform genome analyses. Phylogenetic analysis and divergent time estimation revealed that bla NDM-1 -positive P. aeruginosa ST308 was introduced into Singapore in 2005 (95 % highest posterior density: 2001 to 2008). Core genome, resistome, and analyses of all local bla NDM-1 -positive ST308 isolates showed chromosomal integration of multiple antibiotic resistance genes (ARGs) [ aac(3)-Id , aac(6')-Il , aadA6 , aadA11 , dfrB5 , msr ( E ), floR , sul2 , and qnrVC1 ], which was absent in global bla NDM-1 -negative ST308 sequences. Most ARGs and virulence genes were conserved across isolates originating from the three different local hospitals. Close genetic relatedness of the bla NDM-1 -positive ST308 clinical and environmental isolates suggests cocirculation between the hospital environment and human hosts with the hospital environment as a potential reservoir. Core genome single nucleotide polymorphism analyses revealed possible clonal transmission of bla NDM-1 -positive ST308 isolates between the three hospitals over 7 years. Bloodstream isolates accounted for six of 95 (6.3%) clinical isolates. This study reports the introduction of a pathogenic bla NDM-1 -positive P. aeruginosa ST308 more than a decade ago in Singapore and warrants surveillance for wider dissemination. IMPORTANCE P. aeruginosa is a Gram-negative opportunistic pathogen ubiquitously found in the environment and a major cause of nosocomial infections. While the P. aeruginosa ST308 clone has been known to bear bla IMP and bla VIM among global isolates, reports of bla NDM-1 -positive P. aeruginosa ST308 are rare. The local bla NDM-1 -positive P. aeruginosa ST308 isolates detected in this study appear to be unique to this region, with evidence of chromosomal acquisition of multiple ARGs compared to global bla NDM-1 -negative P. aeruginosa ST308 isolates. Surveillance in Singapore and beyond for dissemination is essential to determine whether existing measures are sufficient to control the spread of this ST308 clone., Competing Interests: The authors declare no conflict of interest.

Melatonin, a widely applied cosmetic active ingredient, has a variety of uses as a skin protector through antioxidant and anti-inflammatory functions as well as giving the body UV-induced defenses and immune system support. In the body, melatonin is synthesized from a tryptophan amino acid in a cascade of reactions, but as melatonin is toxic at high concentrations, it is metabolized in the human skin by the cytochrome P450 enzymes. The P450s are diverse heme-based mono-oxygenases that catalyze oxygen atom-transfer processes that trigger metabolism and detoxification reactions in the body. In the catalytic cycle of the P450s, a short-lived high-valent iron(IV)-oxo heme cation radical is formed that has been proposed to be the active oxidant. How and why it activates melatonin in the human body and what the origin of the product distributions is, are unknown. This encouraged us to do a detailed computational study on a typical human P450 isozyme, namely CYP1A1. We initially did a series of molecular dynamics simulations with substrate docked into several orientations. These simulations reveal a number of stable substrate-bound positions in the active site, which may lead to differences in substrate activation channels. Using tunneling analysis on the full protein structures, we show that two of the four binding conformations lead to open substrate-binding pockets. As a result, in these open pockets, the substrate is not tightly bound and can escape back into the solution. In the closed conformations, in contrast, the substrate is mainly oriented with the methoxy group pointing toward the heme, although under a different angle. We then created large quantum cluster models of the enzyme and focused on the chemical reaction mechanisms for melatonin activation, leading to competitive O-demethylation and C 6 -aromatic hydroxylation pathways. The calculations show that active site positioning determines the product distributions, but the bond that is activated is not necessarily closest to the heme in the enzyme-substrate complex. As such, the docking and molecular dynamics positioning of the substrate versus oxidant can give misleading predictions on product distributions. In particular, in quantum mechanics cluster model I, we observe that through a tight hydrogen bonding network, a preferential 6-hydroxylation of melatonin is obtained. However, O-demethylation becomes possible in alternative substrate-binding orientations that have the C 6 -aromatic ring position shielded. Finally, we investigated enzymatic and non-enzymatic O-demethylation processes and show that the hydrogen bonding network in the substrate-binding pocket can assist and perform this step prior to product release from the enzyme.

Importance: Assessing booster effectiveness of COVID-19 mRNA vaccine and inactivated SARS-CoV-2 vaccine over longer time intervals and in response to any further SARS-CoV-2 variants is crucial in determining optimal COVID-19 vaccination strategies., Objective: To determine levels of protection against severe COVID-19 and confirmed SARS-CoV-2 infection by types and combinations of vaccine boosters in Singapore during the Omicron wave., Design, Setting, and Participants: This cohort study included Singapore residents aged 30 years or more vaccinated with either at least 2 doses of mRNA COVID-19 vaccines (ie, Pfizer-BioNTech BNT162b2 or Moderna mRNA-1273) or inactivated SARS-CoV-2 vaccines (Sinovac CoronaVac or Sinopharm BBIBP-CorV) as of March 10, 2022. Individuals with a known SARS-CoV-2 infection prior to December 27, 2021, an infection on or before the date of their second vaccine dose, or with reinfection cases were excluded., Exposures: Two or 3 doses of Pfizer-BioNTech BNT162b2, Moderna mRNA-1273, Sinovac CoronaVac, or Sinopharm BBIBP-CorV., Main Outcomes and Measures: Notified infections from December 27, 2021, to March 10, 2022, adjusted for age, sex, race, housing status, and calendar days. Estimated booster effectiveness, defined as the relative incidence-rate reduction of severe disease (supplemental oxygen, intensive care, or death) or confirmed infection following 3-dose vaccination compared with 5 months after second mRNA dose, was determined using binomial regression., Results: Among 2 441 581 eligible individuals (1 279 047 [52.4%] women, 846 110 (34.7%) aged 60 years and older), there were 319 943 (13.1%) confirmed SARS-CoV-2 infections, of which 1513 (0.4%) were severe COVID-19 cases. mRNA booster effectiveness against confirmed infection 15 to 60 days after boosting was estimated to range from 31.7% to 41.3% for the 4 boosting combinations (homologous BNT162b2, homologous mRNA-1273, 2-dose BNT162b2/mRNA-1273 booster, and 2-dose mRNA-1273/BNT162b2 booster). Five months and more after boosting, estimated booster effectiveness against confirmed infection waned, ranging from -2.8% to 14.6%. Against severe COVID-19, estimated mRNA booster effectiveness was 87.4% (95% CI, 83.3%-90.5%) 15 to 60 days after boosting and 87.2% (95% CI, 84.2%-89.7%) 5 to 6 months after boosting, with no significant difference comparing vaccine combinations. Booster effectiveness against severe COVID-19 15 days to 330 days after 3-dose inactivated COVID-19 vaccination, regardless of combination, was estimated to be 69.6% (95% CI, 48.7%-81.9%)., Conclusions and Relevance: Booster mRNA vaccine protection against severe COVID-19 was estimated to be durable over 6 months. Three-dose inactivated SARS-CoV-2 vaccination provided greater protection than 2-dose but weaker protection compared with 3-dose mRNA.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo , VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yeo, Chong, Chen, Lim, Ng, Yan, Chu, Chow and Alonso.)

Objective: To evaluate the quality of Arnebiae Radix (AR) and Dictamni Cortex (DC) and study the efficacy of herbal extracts of these two herbs on the treatment of allergic contact dermatitis (ACD)., Methods: Qualitative and quantitative analysis of effective components was performed using High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), and HPLC-Quadrupole Time of Flight-Mass Spectrometry (HPLC-QTOF-MS). In vitro allergic ACD 3D model was established by incubating 3D reconstructed human epidermis (RHE) with skin sensitizer, potassium dichromate. A total of 65 gene expression that were associated with ACD, which included 24 antioxidant responsive element (ARE) and 41 SENS-IS genes were quantified by qRT-PCR. More than or equal to 10 ARE genes and 18 SENN-IS genes were induced by 1.3-fold, demonstrating the successful establishment of in vitro ACD model. Oil extracts of AR and DC were applied on the in vitro ACD model to study the efficacy., Results: Batch 3 of AR and batch 2 of DC showed presence of all active ingredients with the highest concentrations. Active ingredients of the herbs were extracted using a special oil and formulated into herbal oil extracts. The herbal oil extracts were able to down regulate the induced genes in the in-vitro ACD skin model, bringing the tissue back to homeostatic status., Conclusion: The oil extracts showed the potent efficacy of using AR and DC in ACD treatment. The combination study will be done to optimize the formulation ratio which will be developed into a topical cream., Competing Interests: 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., (© 2021 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V.)

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71., (© 2021 The Authors.)

Introduction : Hand, foot and mouth disease (HFMD) is a serious public health concern in the Asia-Pacific region with recurrent cyclical outbreaks. Enterovirus 71 (EV-A71) and coxsackievirus type A are the main causative agents of HFMD. While majority of HFMD cases are mild and self-limiting, neurological complications have been reported for EV-A71 associated HFMD. There is currently no effective treatment against HFMD and monovalent vaccines against EV-A71 are currently limited to the Chinese market. Areas covered : As of today, HFMD antiviral development has focused on EV-A71 and involves conventional screening of drug libraries. In recent years, attention has shifted toward identifying druggable host factors to avoid drug resistance and identify drug candidates with broader antiviral activity across EV-A71 genogroups and other HFMD causative agents. Expert opinion : The effective development of HFMD interventions requires us to address the gaps in our understanding of its pathogenesis at the molecular level. The limited resources devoted to the development of HFMD treatment strategies worldwide also contribute to the slow progress of promising drug and vaccine candidates along the clinical pipeline.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.