Severe Neurological Disease Research Articles

Efforts to Reduce Headache in Grade II Tuberculosis Meningitis Patients: A Case Study

Tuberculous meningitis is a life-threatening disease and can cause severe manifestations and neurological disorders. Headache is one of the symptoms that occurs in patients with tuberculosis meningitis. An 18-year-old woman was admitted with complaints of unilateral headache in the holocranial region with a description of throbbing pain on a scale of 6 (0-10), which decreases when she closes her eyes and sleeps. The pain is felt continuously and slightly interferes with activities. This case study aim to describe intervention that can reduce headache in Grade II Tuburculosis Meningitis. Nursing actions are given to minimize the pain experienced by patients with tuberculosis meningitis, namely carrying out pharmacological and non-pharmacological pain management by collaborating in giving paracetamol 500 mg and teaching non-pharmacological pain management by facilitating patient rest, teaching deep breathing relaxation. In addition, immediate treatment is needed by administering anti-tuberculosis drugs (OAT) and corticosteroids. Corticosteroids are given to minimize complications and death rates by suppressing the inflammatory response in the subarachnoid space. After being given care, the problem of acute pain is partially resolved so that further treatment is needed with participation of the patient and family for the success of nursing care. It is necessary to improve pain management in treating patients with TB meningitis headaches.

Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Epigenetic regulation and factors that influence the effect of iPSCs-derived neural stem/progenitor cells (NS/PCs) in the treatment of spinal cord injury.

Spinal cord injury (SCI) is a severe neurological disorder that causes neurological impairment and disability. Neural stem/progenitor cells (NS/PCs) derived from induced pluripotent stem cells (iPSCs) represent a promising cell therapy strategy for spinal cord regeneration and repair. However, iPSC-derived NS/PCs face many challenges and issues in SCI therapy; one of the most significant challenges is epigenetic regulation and that factors that influence this mechanism. Epigenetics refers to the regulation of gene expression and function by DNA methylation, histone modification, and chromatin structure without changing the DNA sequence. Previous research has shown that epigenetics plays a crucial role in the generation, differentiation, and transplantation of iPSCs, and can influence the quality, safety, and outcome of transplanted cells. In this study, we review the effects of epigenetic regulation and various influencing factors on the role of iPSC-derived NS/PCs in SCI therapy at multiple levels, including epigenetic reprogramming, regulation, and the adaptation of iPSCs during generation, differentiation, and transplantation, as well as the impact of other therapeutic tools (e.g., drugs, electrical stimulation, and scaffolds) on the epigenetic status of transplanted cells. We summarize our main findings and insights in this field and identify future challenges and directions that need to be addressed and explored.

Signatures of migraine aura in high-density-EEG

ObjectiveCortical spreading depolarization is highly conserved among the species. It is easily detectable in direct cortical surface recordings and has been recorded in the cortex of humans with severe neurological disease. It is considered the pathophysiological correlate of human migraine aura, but direct electrophysiological evidence is still missing. As signatures of cortical spreading depolarization have been recognized in scalp EEG, we investigated typical spontaneous migraine aura, using full band high-density EEG (HD-EEG). MethodsIn this prospective study, patients with migraine with aura were investigated during spontaneous migraine aura and interictally. Time compressed HD-EEG were analyzed for the presence of cortical spreading depolarization characterized by (a) slow potential changes below 0.05 Hz, (b) suppression of faster activity from 0.5 Hz − 45 Hz (c) spreading of these changes to neighboring regions during the aura phase. Further, topographical changes in alpha-power spectral density (8–14 Hz) during aura were analyzed. ResultsIn total, 26 HD-EEGs were recorded in patients with migraine with aura, thereof 10 HD-EEGs during aura. Eight HD-EEGs were recorded in the same subject. During aura, no slow potentials were recorded, but alpha-power was significantly decreased in parieto-occipito-temporal location on the hemisphere contralateral to visual aura, lasting into the headache phase. Interictal alpha-power in patients with migraine with aura did not differ significantly from age- and sex-matched healthy controls. ConclusionsUnequivocal signatures of spreading depolarization were not recorded with EEG on the intact scalp in migraine. The decrease in alpha-power contralateral to predominant visual symptoms is consistent with focal depression of spontaneous brain activity as a consequence of cortical spreading depolarization but is not specific thereof. SignificanceCortical spreading depolarization is relevant in migraine, other paroxysmal neurological disorders and neurointensive care.

Comparing the efficacy of anti-seizure medications using matched cohorts on a large insurance claims database

Epilepsy is a severe chronic neurological disease affecting 60 million people worldwide. Primary treatment is with anti-seizure medicines (ASMs), but many patients continue to experience seizures. We used retrospective insurance claims data on 280,587 patients with uncontrolled epilepsy (UE), defined as status epilepticus, need for a rescue medicine, or admission or emergency visit for an epilepsy code. We conducted a computational risk ratio analysis between pairs of ASMs using a causal inference method, in order to match 1034 clinical factors and simulate randomization. Data was extracted from the MarketScan insurance claims Research Database records from 2011 to 2015.The cohort consisted of individuals over 18 years old with a diagnosis of epilepsy who took one of eight ASMs and had more than a year of history prior to the filling of the drug prescription. Seven ASM exposures were analyzed: topiramate, phenytoin, levetiracetam, gabapentin, lamotrigine, valproate, and carbamazepine or oxcarbazepine (treated as the same exposure).We calculated the risk ratio of UE between pairs of ASM after controlling for bias with inverse propensity weighting applied to 1034 factors, such as demographics, confounding illnesses, non-epileptic conditions treated by ASMs, etc. All ASMs exhibited a significant reduction in the prevalence of UE, but three drugs showed pair-wise differences compared to other ASMs. Topiramate consistently was associated with a lower risk of UE, with a mean risk ratio range of 0.68–0.93 (average 0.82, CI: 0.56–1.08). Phenytoin and levetiracetam were consistently associated with a higher risk of UE with mean risk ratio ranges of 1.11 to 1.47 (average 1.13, CI 0.98–1.65) and 1.15 to 1.43 (average 1.2, CI 0.72–1.69), respectively.Large-scale retrospective insurance claims data - combined with causal inference analysis - provides an opportunity to compare the effect of treatments in real-world data in populations 1,000-fold larger than those in typical randomized trials. Our causal analysis identified the clinically unexpected finding of topiramate as being associated with a lower risk of UE; and phenytoin and levetiracetam as associated with a higher risk of UE (compared to other studied drugs, not to baseline). However, we note that our data set for this study only used insurance claims events, which does not comprise actual seizure frequencies, nor a clear picture of side effects. Our results do not advocate for any change in practice but demonstrate that conclusions from large databases may differ from and supplement those of randomized trials and clinical practice and therefore may guide further investigation.

A Drosophila model of mitochondrial disease phenotypic heterogeneity.

Mutations in genes that affect mitochondrial function cause primary mitochondrial diseases. Mitochondrial diseases are highly heterogeneous and even patients with the same mitochondrial disease can exhibit broad phenotypic heterogeneity, which is poorly understood. Mutations in subunits of mitochondrial respiratory complex I cause complex I deficiency, which can result in severe neurological symptoms and death in infancy. However, some complex I deficiency patients present with much milder symptoms. The most common nuclear gene mutated in complex I deficiency is the highly conserved core subunit NDUFS1. To model the phenotypic heterogeneity in complex I deficiency, we used RNAi lines targeting the Drosophila NDUFS1 homolog ND-75 with different efficiencies. Strong knockdown of ND-75 in Drosophila neurons resulted in severe behavioural phenotypes, reduced lifespan, altered mitochondrial morphology, reduced endoplasmic reticulum (ER)-mitochondria contacts and activation of the unfolded protein response (UPR). By contrast, weak ND-75 knockdown caused much milder behavioural phenotypes and changes in mitochondrial morphology. Moreover, weak ND-75 did not alter ER-mitochondria contacts or activate the UPR. Weak and strong ND-75 knockdown resulted in overlapping but distinct transcriptional responses in the brain, with weak knockdown specifically affecting proteosome activity and immune response genes. Metabolism was also differentially affected by weak and strong ND-75 knockdown including gamma-aminobutyric acid (GABA) levels, which may contribute to neuronal dysfunction in ND-75 knockdown flies. Several metabolic processes were only affected by strong ND-75 knockdown including the pentose phosphate pathway and the metabolite 2-hydroxyglutarate (2-HG), suggesting 2-HG as a candidate biomarker of severe neurological mitochondrial disease. Thus, our Drosophila model provides the means to dissect the mechanisms underlying phenotypic heterogeneity in mitochondrial disease.

Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children.

The past decade has seen the global reemergence and rapid spread of enterovirus D68 (EV-D68), a respiratory pathogen that causes severe respiratory illness and paralysis in children. EV-D68 was first isolated in 1962 from children with pneumonia. Sporadic cases and small outbreaks have been reported since then with a major respiratory disease outbreak in 2014 associated with an increased number of children diagnosed with polio-like paralysis. From 2014-2018, major outbreaks have been reported every other year in a biennial pattern with > 90% of the cases occurring in children under the age of 16. With the outbreak of SARS-CoV-2 and the subsequent COVID-19 pandemic, there was a significant decrease in the prevalence EV-D68 cases along with other respiratory diseases. However, since the relaxation of pandemic social distancing protocols and masking mandates the number of EV-D68 cases have begun to rise again - culminating in another outbreak in 2022. Here we review the virology, pathogenesis, and the immune response to EV-D68, and discuss the epidemiology of EV-D68 infections and the divergence of contemporary strains from historical strains. Finally, we highlight some of the key challenges in the field that remain to be addressed.

Clinical epidemiology, determinants, and outcomes of viral encephalitis in Ghana; a cross-sectional study.

Viral encephalitis is a rare, yet severe neurological disorder. It poses a significant public health threat due to its high morbidity and mortality. Despite the disproportionate burden of the disease in impoverished African countries, the true extent of the problem remains elusive due to the scarcity of accurate diagnostic methods. The absence of timely and effective diagnostic tools, particularly Real-time Polymerase Chain Reaction, has led to misguided treatment, and an underestimation of the disease burden in Ghana. We conducted a prospective cross-sectional study to determine the viral aetiologies of encephalitis among patients presenting to a major referral hospital in Ghana from May 2019 and August 2022. The study aimed at providing a comprehensive information on the clinical epidemiology, and outcomes of viral encephalitis in Ghana. Clinical samples were collected from patients presenting with signs and symptoms of encephalitis and tested for viral agents using real-time polymerase chain reaction. We assessed the clinical epidemiology, risk factors and outcome of individuals using descriptive and logistic regression analysis. Seventy-seven (77) patients were enrolled unto the study. The participants frequently presented with fever (85.7%), seizures (80.5%), lethargy (64.9%) and headache (50.6%). Viruses were detected in 40.3% of the study participants in either cerebrospinal fluid, rectal or oral swab samples. The most frequently detected viruses were cytomegalovirus (48.4%), enteroviruses (38.7%) and HSV (29.0%). Twenty-one (27.3%) of the patients died while on hospital admission. Gender (OR = 5.70 (1.536-1.172), p = 0.01), and negative polymerase chain reaction test results were identified as significant factors associated with death. Antiviral treatment increased the chance of survival of viral encephalitis patients by 21.8%. Our results validate the crucial role of molecular tools as essential for the rapid diagnosis of viral encephalitis, enabling effective treatment and improved patient outcomes. This study contributes valuable epidemiological and clinical insight into viral encephalitis in Ghana.

Identification of the pyridoxal 5'-phosphate allosteric site in human pyridox(am)ine 5'-phosphate oxidase.

Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.

Covert consciousness.

Covert consciousness is a state of residual awareness following severe brain injury or neurological disorder that evades routine bedside behavioral detection. Patients with covert consciousness have preserved awareness but are incapable of self-expression through ordinary means of behavior or communication. Growing recognition of the limitations of bedside neurobehavioral examination in reliably detecting consciousness, along with advances in neurotechnologies capable of detecting brain states or subtle signs indicative of consciousness not discernible by routine examination, carry promise to transform approaches to classifying, diagnosing, prognosticating and treating disorders of consciousness. Here we describe and critically evaluate the evolving clinical category of covert consciousness, including approaches to its diagnosis through neuroimaging, electrophysiology, and novel behavioral tools, its prognostic relevance, and open questions pertaining to optimal clinical management of patients with covert consciousness recovering from severe brain injury.

Identification of Senescence-Related Biomarkers and Regulatory Networks in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with substantial societal implications. Cellular senescence plays a critical role in ICH pathogenesis. This study aims to identify senescence-related biomarkers in ICH for diagnostic and therapeutic purposes. Raw data from GSE24265 in Gene Expression Omnibus was downloaded. Senescence-related genes were acquired from CellAge. Differential gene analysis was done between patients with ICH and controls. The intersection of ICH differentially expressed genes and senescence-related genes for senescence-related ICH genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Protein-protein interaction network was constructed through the Search Tool for the Retrieval of Interacting Genes. Single sample gene set enrichment analysis was done for immune cell infiltration and function evaluation in control and ICH groups. miRWalk2.0 database was used for microRNA predictions targeting ICH biomarkers. Transcriptional regulatory relationships unraveled by sentence-based text mining database was employed to predict transcription factors regulating identified biomarkers. Thirteen senescence-related ICH genes were identified. They were primarily enriched in the positive regulation of angiogenesis and the Advanced Glycation End Product -Receptor for AGE signaling pathway in diabetic complications. Validation in the GSE149317 data set and receiver operating characteristic analysis highlighted Caveolin 1, C-X-C Motif Chemokine Ligand 1, ETS proto-oncogene 1, transcription factor, and Serpin Family E Member 1 as potential ICH biomarkers. Single sample gene set enrichment analysis revealed increased Type 2 T helper cell 2_cells, Treg cells, and immune functions like Antigen-presenting cells_co_stimulation in patients with ICH. Fourteen microRNA, including has-miR-6728-3p, were predicted to regulate these biomarkers. transcription factors such as PPARG, RARA, HMGA1, and NFKB1 were identified as potential regulators of the ICH biomarkers. Caveolin 1, C-X-C Motif Chemokine Ligand 1, ETS proto-oncogene 1, transcription factor, and Serpin Family E Member 1 may serve as valuable biomarkers in ICH. Targeting these genes could contribute to ICH prevention and treatment.

Evaluation of Microbiological quality and antibacterial activity of Cannabis artisanal oil used in Brazil

The medicinal use of Cannabis sativa has been highlighted as an alternative in the treatment of neurological diseases, due to the cannabidiol (CBD), with anticonvulsant properties. This study evaluated the microbiological quality and antibacterial activity of Cannabis extracts used by patients with refractory epilepsy in order to bring more safety in their use and prescription. Around of 70 samples of Cannabis extracts were evaluated, 10 of them used for antibacterial activity tests. Microbiological quality was measured by observing the growth of bacteria, after the inoculation of the Cannabis extract into BHI Agar, BEM Agar, MacConkey Agar and Mueller-Hinton Agar culture media. For antimicrobial activity, a modified Kirby-Bauer method was performed with 11 bacterial isolates considered potentially pathogenic. Of the analyzed samples, 70 showed no bacterial contamination during its preparation, distribution and storage process, making them safe from a microbiological perspective for human consumption. Regarding antimicrobial activity, the 10 samples tested showed no inhibitory activity against the listed bacteria. The product may not interfere with the microbiota of users being treated, as some of the isolates are part of the human microbiota. Analysis of the microbiological quality and antibacterial activity of cannabis used in patients with severe neurological disorder is of fundamental importance to ensure better safety in its use. Therefore, all Cannabis extracts tested are free of microbial contamination that may compromise product quality, and none of the extracts used in this study inhibited the in vitro growth of the tested bacteria.

Patient selection considerations for AADC deficiency gene therapy

AbstractBackgroundAromatic ʟ‐amino acid decarboxylase (AADC) deficiency is a rare, severe neurological disorder caused by pathogenic variants in the dopa decarboxylase (DDC) gene, resulting in a combined deficiency of monoamine neurotransmitters. Clinically, patients present with a range of dysfunctions that impact motor, autonomic, and cognitive development. The constellation of symptoms of AADC deficiency varies among patients, and clinical presentation falls across a wide spectrum. However, most patients with AADC deficiency experience significant impairments when compared with children with normal development, irrespective of genotype, phenotype, or disease severity. Further, AADC deficiency is associated with increased mortality.MethodsIn response to the recent approval of a disease‐modifying gene therapy for AADC deficiency, this review presents considerations for the selection of patients for treatment.ConclusionSuggested clinical criteria to determine whether a patient is a candidate for gene therapy are: (1) genetically and biochemically confirmed AADC deficiency; (2) lack of achievement of gross motor milestones and/or persistence of clinically significant movement disorders; (3) persistent neurocognitive or systemic symptoms secondary to AADC deficiency despite standard medical therapy; and (4) informed parental/guardian decision and consent to treatment.

Automatic detection of epileptic seizure using machine learning-based IANFIS-LightGBM system

A sudden increase in electrical activity in the brain is a defining feature of one of the severe neurological diseases known as epilepsy. This abnormality appears as a seizure, and identifying seizures is an important field of research. An essential technique for examining the features of neurological issues brain activities, and epileptic seizures is electroencephalography (EEG). In EEG data, analyzing epileptic irregularities visually requires a lot of time from neurologists. For accurate detection of epileptic seizures, numerous scientific techniques have been used with EEG data, and most of these techniques have produced promising results. For EEG signal classification with a high classification accuracy rate, the present research proposes an enhanced machine learning-based epileptic seizure detection model. The present research provides a hybrid Improved Adaptive Neuro-Fuzzy Inference System (IANFIS)-Light Gradient Boosting Machine (LightGBM) technique for automatically detecting and diagnosing epilepsy from EEG data. The experimental findings were supported by EEG records made available by the German University of Bonn and scalp EEG data acquired at Children’s Hospital Boston. The suggested IANFIS-LightGBM, according to the results, offers the most significant classification accuracy ratings in both situations.

The BONSAI (Brain and Optic Nerve Study with Artificial Intelligence) deep learning system can accurately identify pediatric papilledema on standard ocular fundus photographs

BackgroundPediatric papilledema often reflects an underlying severe neurologic disorder and may be difficult to appreciate, especially in young children. Ocular fundus photographs are easy to obtain even in young children and in nonophthalmology settings. The aim of our study was to ascertain whether an improved deep-learning system (DLS), previously validated in adults, can accurately identify papilledema and other optic disk abnormalities in children. MethodsThe DLS was tested on mydriatic fundus photographs obtained in a multiethnic pediatric population (<17 years) from three centers (Atlanta-USA; Bucharest-Romania; Singapore). The DLS’s multiclass classification accuracy (ie, normal optic disk, papilledema, disks with other abnormality) was calculated, and the DLS’s performance to specifically detect papilledema and normal disks was evaluated in a one-vs-rest strategy using the AUC, sensitivity and specificity, with reference to expert neuro-ophthalmologists. ResultsExternal testing was performed on 898 fundus photographs:447 patients; mean age, 10.33 (231 patients ≤10 years of age; 216, 11-16 years); 558 normal disks, 254 papilledema, 86 other disk abnormalities. Overall multiclass accuracy of the DLS was 89.6% (range, 87.8%-91.6%). The DLS successfully distinguished “normal” from “abnormal” optic disks (AUC 0.99 [0.98-0.99]; sensitivity, 87.3% [84.9%-89.8%]; specificity, 98.5% [97.6%-99.6%]), and “papilledema” from “normal and other” (AUC 0.99 [0.98-1.0]; sensitivity, 98.0% [96.8%-99.4%]; specificity, 94.1% (92.4%-95.9%)]. ConclusionsOur DLS reliably distinguished papilledema from normal optic disks and other disk abnormalities in children, suggesting it could be utilized as a diagnostic aid for the assessment of optic nerve head appearance in the pediatric age group.

Trends in the Utilization of Surgical Modalities for the Treatment of Drug-Resistant Epilepsy: A Comprehensive 10-Year Analysis Using the National Inpatient Sample.

Epilepsy is considered one of the most prevalent and severe chronic neurological disorders worldwide. Our study aims to analyze the national trends in different treatment modalities for individuals with drug-resistant epilepsy and investigate the outcomes associated with these procedural trends in the United States. Using the National Inpatient Sample database from 2010 to 2020, patients with drug-resistant focal epilepsy who underwent laser interstitial thermal therapy (LITT), open surgical resection, vagus nerve stimulation (VNS), or responsive neurostimulation (RNS) were identified. Trend analysis was performed using piecewise joinpoint regression. Propensity score matching was used to compare outcomes between 10 years prepandemic before 2020 and the first peak of the COVID-19 pandemic. This study analyzed a total of 33 969 patients with a diagnosis of drug-resistant epilepsy, with 3343 patients receiving surgical resection (78%), VNS (8.21%), RNS (8%), and LITT (6%). Between 2010 and 2020, there was an increase in the use of invasive electroencephalography monitoring for seizure zone localization (P = .003). There was an increase in the use of LITT and RNS (P < .001), while the use of surgical resection and VNS decreased over time (P < .001). Most of these patients (89%) were treated during the pre-COVID pandemic era (2010-2019), while a minority (11%) underwent treatment during the COVID pandemic (2020). After propensity score matching, the rate of pulmonary complications, postprocedural hematoma formation, and mortality were slightly higher during the pandemic compared with the prepandemic period (P = .045, P = .033, and P = .026, respectively). This study indicates a relative decrease in the use of surgical resections, as a treatment for drug-resistant focal epilepsy. By contrast, newer, minimally invasive surgical approaches including LITT and RNS showed gradual increases in usage.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

BackgroundLesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells.MethodsWe prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts.ResultsLND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND.ConclusionsA complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Development of a luminescence-based method for measuring West Nile Virus MTase activity and its application to screen for antivirals

West Nile virus (WNV) is a flavivirus responsible for causing febrile illness and severe neurological diseases, with an increasing impact on human health around the world. However, there is still no adequate therapeutic treatment available to struggle WNV infections. Therefore, there is an urgent need to develop new techniques to accelerate the discovery of drugs against this pathogen. The main protein implicated in the replication of WNV is the non-structural protein 5 (NS5). This multifunctional protein contains methyltransferase (MTase) activity involved in the capping formation at the 5′-end of RNA and the methylation of internal viral RNA residues, both functions being essential for viral processes, such as RNA translation and escape from the innate immune response.We have developed a straightforward luminescence-based assay to monitor the MTase activity of the WNV NS5 protein with potential for high-throughput screening. We have validated this method as a sensitive and suitable assay for the identification of WNV MTase inhibitors assessing the inhibitory effect of the broad MTase inhibitor sinefungin, a natural nucleoside analog of the universal methyl donor S-adenosyl methionine (SAM). The screening of a small series of purine derivatives identified an adenosine derivative as a dose-dependent inhibitor of the MTase activity. The antiviral efficacy of this compound was further confirmed in WNV infections, displaying a measurable antiviral effect. This result supports the utility of this novel method for the screening of inhibitors against WNV MTase activity, which can be of special relevance to the discovery and development of therapeutics against WNV.

Discovery and structure-activity relationship study of novel isoxazole-based small molecules targeting Zika virus infections.

The Zika virus (ZIKV), a significant public health threat, is transmitted by Aedes aegypti mosquitoes and is associated with severe neurological disorders, particularly in newborns. Currently, there are no approved vaccines or specific therapeutics for ZIKV. Our study focuses on the identification and optimization of isoxazole-based small molecules, specifically through the structural modification of KR-26827, to combat ZIKV infections. Among the synthesized derivatives, 7l emerged as the most promising candidate, showing potent antiviral activity against ZIKV strains and an improved safety profile in vitro. This research underlines the potential of 7l for further development as a ZIKV therapeutic agent.