[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Phenotypic characteristics of myelin oligodendrocyte glycoprotein antibody-associated disease in children: a single-center, retrospective study.

Objective: To analyze the clinical characteristics and follow-up data of children with different clinical phenotypes of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). Methods: The basic demographic and clinical features, laboratory and imaging examination results, and follow-up data of 74 Chinese children with different phenotypes of MOGAD were retrospectively reviewed and analyzed. Results: The male-to-female ratio in this cohort was 1:1.39. The clinical phenotypes of MOGAD included acute disseminated encephalomyelitis (ADEM; n = 37), encephalitis (n = 11), optic neuritis (ON, n = 9), neuromyelitis optica spectrum disorder (NMOSD; n = 9), transverse myelitis (TM; n = 6), leukodystrophy-like manifestations (n = 1), and meningitis (n = 1). The mean age of disease onset was 86 months. The number of leukocytes in the cerebrospinal fluid of patients with ADEM was significantly higher than that in patients with ON but lower than that in patients with TM (p < 0.05). The pathogen detection rate among all patients was 36.5%. Recurrence occurred in 17 patients (23%), with the highest recurrence rate in patients with NMOSD and TM. Patients with recurrence had a significantly higher median age than those without any recurrence (109.00 vs. 82.44 months, p < 0.05). The male-to-female ratio in patients with recurrence was 1:4.67, which differed significantly from that at first onset (p < 0.05). Conclusion: The most common clinical phenotypes of MOGAD in this cohort were ADEM and encephalitis. Recurrence of MOGAD may be related to age and sex, with a higher recurrence rate observed in females. These findings provide a basis for further exploration of the characteristics of different MOGAD phenotypes.

Trpm2 deficiency in microglia attenuates neuroinflammation during epileptogenesis by upregulating autophagy via the AMPK/mTOR pathway.

Epilepsy is one of the most common neurological disorders. Neuroinflammation involving the activation of microglia and astrocytes constitutes an important and common mechanism in epileptogenesis. Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that plays pathological roles in various inflammation-related diseases. Our previous study demonstrated that Trpm2 knockout exhibits therapeutic effects on pilocarpine-induced glial activation and neuroinflammation. However, whether TRPM2 in microglia and astrocytes plays a common pathogenic role in this process and the underlying molecular mechanisms remained undetermined. Here, we demonstrate a previously unknown role for microglial TRPM2 in epileptogenesis. Trpm2 knockout in microglia attenuated kainic acid (KA)-induced glial activation, inflammatory cytokines production and hippocampal paroxysmal discharges, whereas Trpm2 knockout in astrocytes exhibited no significant effects. Furthermore, we discovered that these therapeutic effects were mediated by upregulated autophagy via the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in microglia. Thus, our findings highlight an important deleterious role of microglial TRPM2 in temporal lobe epilepsy.

Targeting nucleolin improves sensitivity to chemotherapy in acute lymphoblastic leukemia.

PURPOSE: Most patients with acute lymphoblastic leukemia (ALL) are treated with chemotherapy as primary care. Although the treatment response is usually positive, resistance and relapse often occur via unknown mechanisms. The purpose of this study was to identify factors associated with chemotherapy resistance in ALL. Here, we present clinical and experimental evidence that overexpression of nucleolin (NCL), a multifunctional nucleolar protein, is linked to drug resistance in ALL. METHODS: NCL mRNA and protein levels were compared between cell lines and patient samples using qRT-PCR and immunoblotting. NCL mRNA levels were compared between patients of different disease stages from our clinic patients' specimens and publicly available ALL patient datasets. Cells and patient-derived xenograft mouse experiments were performed to assess the effect of NCL inhibition on ALL chemotherapy effectiveness. RESULTS: Analysis of patient specimens, and publicly available RNA-sequencing datasets revealed a strong correlation between the abundance of NCL and disease relapse or poor survival in B-ALL. Altering NCL expression results in changes in drug sensitivity in ALL cell lines. High levels of NCL upregulated components of the ATP-binding cassette transporters via activation of the ERK pathway, resulting in a decrease in drug accumulation inside the cells. Targeting NCL with AS1411, an NCL-binding oligonucleotide aptamer, significantly increased the sensitivity of ALL cell lines and cells/patient-derived ALL xenograft mice to chemotherapeutic drugs and prolonged mouse survival. CONCLUSION: Our results highlight NCL as a prognostic marker in B-ALL and a potential therapeutic target to combat chemotherapy resistance in ALL.

A Systematic Review and Meta-Analysis of the R778L Mutation in ATP7B With Wilson Disease in China.

BACKGROUND: Wilson disease (WD) is a hereditary disorder of copper metabolism, caused by mutations in the ATP7B gene. There are more than 1000 pathogenic variants identified in ATP7B. R778L is the most common ATP7B mutation in China. METHODS: To estimate whether R778L is associated with the onset age of WD and other clinical variables. Genotyping results of ATP7B gene were collected in our 22 patients with WD. We then conducted a systematic review and meta-analysis in databases, using the keywords Wilson disease and R778L mutation. RESULTS: After the screening, a total of 23 studies were included, including 3007 patients with WD. Patients with R778L mutation presented at an earlier age (standardized mean difference [SMD] = -0.18 [95% confidence interval, -0.28 to 0.08], P = 0.0004) and had lower ceruloplasmin concentration (SMD = -0.21 [95% confidence interval, -0.40 to -0.02], P = 0.03) than the patients without the R778L mutation. However, sex (odds ratio [OR] = 1.07 [95% confidence interval, 0.89 to 1.29], P = 0.32) and first presentation were not associated with R778L mutation in WD (hepatic: OR = 1.37 [95% confidence interval, 0.87 to 2.16, P = 0.17; neurological: OR = 0.79 [95% confidence interval, 0.48 to 1.30, P = 0.35; mix: OR = 1.04 [95% confidence interval, 0.42 to 2.53, P = 0.87; asymptomatic/others: OR = 1.98 [95% confidence interval, 0.49 to 7.96, P = 0.34). CONCLUSIONS: Our results indicated that the R778L mutation is associated with an earlier presentation and lower ceruloplasmin concentration in China.

Metagenomic next-generation sequencing and proteomics analysis in pediatric viral encephalitis and meningitis.

Introduction: Early and accurate identification of pathogens is essential for improved outcomes in patients with viral encephalitis (VE) and/or viral meningitis (VM). Methods: In our research, Metagenomic next-generation sequencing (mNGS) which can identify viral pathogens unbiasedly was performed on RNA and DNA to identify potential pathogens in cerebrospinal fluid (CSF) samples from 50 pediatric patients with suspected VEs and/or VMs. Then we performed proteomics analysis on the 14 HEV-positive CSF samples and another 12 CSF samples from health controls (HCs). A supervised partial least squaresdiscriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) model was performed using proteomics data. Results: Ten viruses in 48% patients were identified and the most common pathogen was human enterovirus (HEV) Echo18. 11 proteins overlapping between the top 20 DEPs in terms of P value and FC and the top 20 proteins in PLS-DA VIP lists were acquired. Discussion: Our result showed mNGS has certain advantages on pathogens identification in VE and VM and our research established a foundation to identify diagnosis biomarker candidates of HEV-positive meningitis based on MS-based proteomics analysis, which could also contribute toward investigating the HEV-specific host response patterns.

Case report: Pediatric anti-gamma aminobutyric acid-B receptor encephalitis with benign prognosis.

Objective: To explore the clinical characteristics of pediatric anti-gamma-aminobutyric acid-B receptor (GABABR) encephalitis to enhance the understanding and improve the diagnostic and therapeutic strategies for this disease. Methods: We report a rare case of a female pediatric patient with anti-GABABR encephalitis who was treated at the Children's Hospital of Zhejiang University School of Medicine. Literature search was performed to explore the clinical characteristics of pediatric anti-GABABR encephalitis. Results: The patient exhibited recurrent epileptic seizure, status epilepticus, and psychiatric symptoms at the age of 11 years and 10 months. Anti-GABABR antibodies were positive in cerebrospinal fluid and serum. Brain magnetic resonance imaging (MRI) exhibited abnormal signals in the left hippocampus. Symptoms and abnormality of brain MRI were improved after administration of immunosuppressants, anti-seizure and antipsychotic drugs. Two of pediatric anti-GABABR encephalitis with clinical data were identified through literature search. Analysis of these three cases suggested that the pediatric patients primarily experienced limbic encephalitis, with no tumor incidence. A favorable immunotherapy response was demonstrated with a superior prognosis in all the cases. Conclusions: We reported a pediatric anti-GABABR encephalitis case with early age of onset. Promt autoimmune antibody testing and tumor screening, as well as immunomodulatory treatment immediately after a definitive diagnosis are warranted to improve prognosis.

Loss of MiR-155 Sensitizes FLT3-ITD+AML to Chemotherapy and FLT3 Inhibitors via Glycolysis Blocking by Targeting PIK3R1.

FLT3 tyrosine kinase inhibitors in combination with chemotherapy have shown some success in patients with FLT3 mutations. But a variety of mechanisms have led to the rapid resistance to the treatment. One of the most prominent is the metabolic alteration on aerobic glycolysis. We aim to explore the role of a high expressing microRNA, miR-155, in mediating resistance to chemotherapy and FLT3 inhibitor treatment. The deep sequencing data mining revealed the connection between glycolysis and drug resistance. MV411 cells with miR-155 knockout (KO) not only had increased sensitivity to FLT3 inhibitors but also Adriamycin (ADM) treatment. When combined with glycolysis inhibition the treatment response in MV411 cells further increased. Whereas in miR-155 KO cells, a lower glucose consumption level and lactic acid level were observed, and western blotting showed a decreased expression of key enzymes in glycolysis pathways. A negative correlation between PIK3R1 and miR-155 level can be observed in the sequencing data from FLT3-ITD+ AML patients. Moreover, luciferase reporter assay revealed that the 3'UTR of PIK3R1 mRNA can interact with the seed sequence of miR-155-5p. In conclusion, the loss of miR-155 increased treatment sensitivity to both chemotherapy and FLT3 inhibitors in FLT3-ITD+ AML cells via glycolysis blocking by targeting PIK3R1.

Proteomic profiling of cerebrospinal fluid in pediatric myelin oligodendrocyte glycoprotein antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an autoimmune demyelinating disorder of the central nervous system. METHODS: Extracted proteins from 34 cerebrospinal fluid (CSF) samples [patients with MOGAD (MOG group, n = 12); healthy controls (HC group, n = 12); patients with MOG seronegative and metagenomics next-generation sequencing-negative inflammatory neurological diseases (IND group, n = 10)] were processed and subjected to label-free quantitative proteomics. Supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) models were also performed based on proteomics data. Functional analysis of differentially expressed proteins (DEPs) was performed using Gene Ontology, InterPro, and Kyoto Encyclopedia Genes and Genomes. An enzyme-linked immunosorbent assay was used to determine the complement levels in serum from patients with MOGAD. RESULTS: Four hundred and twenty-nine DEPs (149 upregulated and 280 downregulated proteins) were identified in the MOG group compared to the HC group according to the P value and fold change (FC). Using the O-PLS-DA model, 872 differentially abundant proteins were identified with variable importance projection (VIP) scores > 1. Five proteins (gamma-glutamyl hydrolase, cathepsin F, interalpha-trypsin inhibitor heavy chain 5, latent transforming growth factor beta-binding protein 4 and leukocyte-associated immunoglobulin-like receptor 1) overlapping between the top 30 DEPs with top-ranked P value and FC and top 30 proteins in PLS-DA VIP lists were acquired. Functional analysis revealed that the dysregulated proteins in the MOG group were primarily involved in complement and coagulation cascades, cell adhesion, axon guidance, and glycosphingolipid biosynthesis compared to the HC group. CONCLUSION: The proteomic alterations in CSF samples from children with MOGAD identified in the current study might provide opportunities for developing novel biomarker candidates.

Genetic Knockout of TRPM2 Increases Neuronal Excitability of Hippocampal Neurons by Inhibiting Kv7 Channel in Epilepsy.

Epilepsy is a chronic brain disease that makes serious cognitive and motor retardation. Ion channels affect the occurrence of epilepsy in various ways, but the mechanisms have not yet been fully elucidated. Transient receptor potential melastain2 (TRPM2) ion channel is a non-selective cationic channel that can permeate Ca2+ and critical for epilepsy. Here, TRPM2 gene knockout mice were used to generate a chronic kindling epilepsy model by PTZ administration in mice. We found that TRPM2 knockout mice were more susceptible to epilepsy than WT mice. Furthermore, the neuronal excitability in the hippocampal CA1 region of TRPM2 knockout mice was significantly increased. Compared with WT group, there were no significant differences in the input resistance and after hyperpolarization of CA1 neurons in TRPM2 knockout mice. Firing adaptation rate of hippocampal CA1 pyramidal neurons of TRPM2 knockout mice was lower than that of WT mice. We also found that activation of Kv7 channel by retigabine reduced the firing frequency of action potential in the hippocampal pyramidal neurons of TRPM2 knockout mice. However, inhibiting Kv7 channel increased the firing frequency of action potential in hippocampal pyramidal neurons of WT mice. The data suggest that activation of Kv7 channel can effectively reduce epileptic seizures in TRPM2 knockout mice. We conclude that genetic knockout of TRPM2 in hippocampal CA1 pyramidal neurons may increase neuronal excitability by inhibiting Kv7 channel, affecting the susceptibility to epilepsy. These findings may provide a potential therapeutic target for epilepsy.

Captopril alleviates epilepsy and cognitive impairment by attenuation of C3-mediated inflammation and synaptic phagocytosis.

Evidence from experimental and clinical studies implicates immuno-inflammatory responses as playing an important role in epilepsy-induced brain injury. Captopril, an angiotensin-converting enzyme inhibitor (ACEi), has previously been shown to suppress immuno-inflammatory responses in a variety of neurological diseases. However, the therapeutic potential of captopril on epilepsy remains unclear. In the present study, Sprague Dawley (SD) rats were intraperitoneally subjected to kainic acid (KA) to establish a status epilepticus. Captopril (50 mg/kg, i.p.) was administered daily following the KA administration from day 3 to 49. We found that captopril efficiently suppressed the KA-induced epilepsy, as measured by electroencephalography. Moreover, captopril ameliorated the epilepsy-induced cognitive deficits, with improved performance in the Morris water maze, Y-maze and novel objective test. RNA sequencing (RNA-seq) analysis indicated that captopril reversed a wide range of epilepsy-related biological processes, particularly the glial activation, complement system-mediated phagocytosis and the production of inflammatory factors. Interestingly, captopril suppressed the epilepsy-induced activation and abnormal contact between astrocytes and microglia. Immunohistochemical experiments demonstrated that captopril attenuated microglia-dependent synaptic remodeling presumably through C3-C3ar-mediated phagocytosis in the hippocampus. Finally, the above effects of captopril were partially blocked by an intranasal application of recombinant C3a (1.3 µg/kg/day). Our findings demonstrated that captopril reduced the occurrence of epilepsy and cognitive impairment by attenuation of inflammation and C3-mediated synaptic phagocytosis. This approach can easily be adapted to long-term efficacy and safety in clinical practice.

Mitochondrial DNA variant spectrum and the association with chronic tic disorders.

BACKGROUND AND PURPOSE: Tic disorders (TDs) are childhood onset neuropsychiatric disorders characterized by single or multiple sudden, rapid, recurrent, and motor tics and/or vocal tics. Several nuclear genes that are involved in mitochondrial functions suggest a potential role of mitochondria in TDs. METHODS: To evaluate the association of mitochondrial DNA (mtDNA) variants with TDs, we screened the whole mitochondrial genomes in 493 TD patients and 109 age- and sex-matched healthy controls using next generation sequencing technology. RESULTS: A total of 1918 mtDNA variants including 1220 variants in patients only, 154 variants in controls only, and 544 variants shared by both cases and controls were identified. We found a higher number of overall mtDNA variants in TD patients (p = 0.00028). The variant density in MT-ATP6/8 and MT-CYB coding regions showed a significant difference between TD patients and controls (p = 0.0025 and p = 0.003, respectively). Furthermore, we observed a significant association of 15 common variants with TD based on an additive model, including m.14766C > T, m.14783 T > C, m.14905G > A, and m.15301G > A in MT-CYB; m.4769A > G, m.10398A > G, m.12705C > T, and m.12850A > G in MT-ND genes; m.7028C > T in MT-CO1; m.8701A > G in MT-ATP6; two variants with m.16223C > T, m.5580 T > C in noncoding regions; and three rRNA variants with m.1438A > G and m.750A > G in RNR1, and m.2352 T > C in RNR2. CONCLUSIONS: Our data provide evidence of mtDNA variants associated with TDs. The accumulation of the heteroplasmic levels may increase the risk of TDs. Replication studies with larger samples are necessary to understand the pathogenesis of TDs.

Miconazole exerts disease-modifying effects during epilepsy by suppressing neuroinflammation via NF-κB pathway and iNOS production.

Neuroinflammation contributes to the generation of epilepsy and has been proposed as an effective therapeutic target. Recent studies have uncovered the potential effects of the anti-fungal drug miconazole for treating various brain diseases by suppressing neuroinflammation but have not yet been studied in epilepsy. Here, we investigated the effects of different doses of miconazole (5, 20, 80 mg/kg) on seizure threshold, inflammatory cytokines release, and glial cells activation in the pilocarpine (PILO) pentylenetetrazole (PTZ), and intrahippocampal kainic acid (IHKA) models. We demonstrated that 5 and 20 mg/kg miconazole increased seizure threshold, but only 20 mg/kg miconazole reduced inflammatory cytokines release, glial cells activation, and morphological alteration during the early post-induction period (24 h, 3 days). We further investigated the effects of 20 mg/kg miconazole on epilepsy (4 weeks after KA injection). We found that miconazole significantly attenuated cytokines production, glial cells activation, microglial morphological changes, frequency and duration of recurrent hippocampal paroxysmal discharges (HPDs), and neuronal and synaptic damage in the hippocampus during epilepsy. In addition, miconazole suppressed the KA-induced activation of the NF-κB pathway and iNOS production. Our results indicated miconazole to be an effective drug for disease-modifying effects during epilepsy, which may act by attenuating neuroinflammation through the suppression of NF-κB activation and iNOS production. At appropriate doses, miconazole may be a safe and effective approved drug that can easily be repositioned for clinical practice.

Optical tweezers and Raman spectroscopy for single-cell classification of drug resistance in acute lymphoblastic leukemia.

Laser Tweezers Raman Spectroscopy (LTRS) is a combination of laser tweezers and Raman spectroscopy. It is a physical tool based on the mechanical effects of the laser, which can be used to study single living cells in suspension in a fast and non-destructive way. Our work aims to establish a methodology system based on LTRS to rapidly and non-destructively detect the resistance of acute lymphoblastic leukemia (ALL) cells and to provide a new idea for the evaluation of the resistance of ALL cells. Two specific adriamycin-resistant and parental ALL cells BALL-1 and Nalm6 were included in this study. Adriamycin resistant cells can induce the spectral differences, which can be detected by LTRS initially. To ensure the accuracy of the results, we use the principal components analysis (PCA) as well as the classification and regression trees (CRT) algorithms, which show that the specificity and sensitivity of LTRS are above 90%. In addition, to further clarify the chemoresistance status of ALL cells, we used the CRT models and receiver operating characteristic (ROC) curves which are based on the band data to look for some important bands and band intensity ratios that have strong pointing significance. Our work proves that LTRS analysis combined with multivariate statistical analyses have great potential to be a novel analytical strategy at the single-cell level for rapidly evaluating the chemoresistance status of ALL cells.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Circulating MicroRNAs From Plasma Small Extracellular Vesicles as Potential Diagnostic Biomarkers in Pediatric Epilepsy and Drug-Resistant Epilepsy.

Pediatric epilepsy is a neurological condition that causes repeated and unprovoked seizures and is more common in 1-5-year-old children. Drug resistance has been indicated as a key challenge in improving the clinical outcomes of patients with pediatric epilepsy. In the present study, we aimed to identify plasma small extracellular vesicles (sEVs) derived microRNAs (miRNAs) from the plasma samples of children for predicting the prognosis in patients with epilepsy and drug-resistant epilepsy. A total of 90 children clinically diagnosed with epilepsy [46 antiepileptic drug (AED)-responsive epilepsy and 44 drug-resistant epilepsy] and 37 healthy controls (HCs) were enrolled in this study. RNA sequencing was performed to identify plasma sEVs derived miRNAs isolated from the children's plasma samples. Differentially expressed plasma sEVs derived miRNAs were identified using bioinformatics tools and were further validated by reverse transcription-polymerase chain reaction and receiver operator characteristic (ROC) curve analysis. In the present study, 6 miRNAs (hsa-miR-125b-5p, hsa-miR-150-3p, hsa-miR-199a-3p, hsa-miR-584-5p hsa-miR-199a-5p, and hsa-miR-342-5p) were selected for further validation. hsa-miR-584-5p, hsa-miR-342-5p, and hsa-miR-150-5p with area under curve (AUC) values of 0.846, 0.835, and 0.826, respectively, were identified as promising biomarkers of epilepsy. A logistic model combining three miRNAs (hsa-miR-584-5p, hsa-miR-342-5p, and hsa-miR-199a-3p) could achieve an AUC of 0.883 and a six miRNAs model (hsa-miR-342-5p, hsa-miR-584-5p, hsa-miR-150-5p, hsa-miR-125b-5p, hsa-miR-199a-3p, and hsa-miR-199a-5p) could attain an AUC of 0.888. The predicted probability of multiple miRNA panels was evaluated for differentiating between drug-resistant children and drug-responsive children. The AUC of a six-miRNA panel (hsa-miR-342-5p, hsa-miR-584-5p, hsa-miR-150-5p, hsa-miR-125b-5p, hsa-miR-199a-3p, and hsa-miR-199a-5p) reached 0.823. We identified and confirmed plasma sEVs derived miRNA biomarkers that could be considered as potential therapeutic targets for pediatric epilepsy and drug-resistant epilepsy.