Plasma proteome profiling reveals dynamic of cholesterol marker after dual blocker therapy.

Dual blocker therapy (DBT) has the enhanced antitumor benefits than the monotherapy. Yet, few effective biomarkers are developed to monitor the therapy response. Herein, we investigate the DBT longitudinal plasma proteome profiling including 113 longitudinal samples from 22 patients who received anti-PD1 and anti-CTLA4 DBT therapy. The results show the immune response and cholesterol metabolism are upregulated after the first DBT cycle. Notably, the cholesterol metabolism is activated in the disease non-progressive group (DNP) during the therapy. Correspondingly, the clinical indicator prealbumin (PA), free triiodothyronine (FT3) and triiodothyronine (T3) show significantly positive association with the cholesterol metabolism. Furthermore, by integrating proteome and radiology approach, we observe the high-density lipoprotein partial remodeling are activated in DNP group and identify a candidate biomarker APOC3 that can reflect DBT response. Above, we establish a machine learning model to predict the DBT response and the model performance is validated by an independent cohort with balanced accuracy is 0.96. Thus, the plasma proteome profiling strategy evaluates the alteration of cholesterol metabolism and identifies a panel of biomarkers in DBT.

Impacts of mixed ferrous sulfate-biochar additives on humification and bacterial community during electric field-assisted aerobic composting.

This study assessed the impact of nine mixed ferrous sulfates and biochars on electric field-assisted aerobic composting (EAC), focusing on the spectroscopy of dissolved organic matter (DOM) and microbial communities. Adding 1.05 % ferrous sulfate and 5.25 % biochar to EAC increased the specific ultraviolet absorbances at 254 and 280 nm by 142.3 % and 133.9 % on day 35, respectively. This ratio accelerated the early response of carboxyl groups (-COOH) and lignin (C = C), enhancing the relative abundance of Thermobifida (4.0 %) and Thermopolyspora (4.3 %). The condition contributed to humus precursor formation on day 5, increasing the maximum fluorescence intensity of the humus-like component by 74.2 % compared to the control on day 35. This study is the first to develop a combined and efficient organic and inorganic additive by multiple-variable experimentation for DOM humification. Consequently, it optimizes EAC for solid waste recycling.

Influencing Factors of Generic Prescribing Behavior of Physicians: A Structural Equation Model Based on the Theory of Planned Behavior.

Background: Although affordable generics could probably contribute to the solution of rapidly increasing pharmaceutical expenditure, those drugs are prescribed at a lower rate in China. Physicians' perception and knowledge of generics have a great influence on their prescribing behavior. Objective: This study aimed to identify factors that affect physicians' generic prescribing behavior based on the theory of planned behaviors (TPB). Methods: Data were collected by both electronic and paper-based surveys from 1297 Chinese physicians, and 1047 surveys were retained. The structural equation model (SEM) was employed to investigate the relationship between four behavioral constructs, namely, attitudes, subjective norms, perceived control of behaviors, and intentions. Results: About 50% of Chinese physicians had a positive attitude towards generic drugs that had passed the "Consistency Evaluation of Quality and Efficacy of Generic Drugs" (high-quality generic drugs), but their knowledge of generic drugs was relatively inadequate. The path coefficients for the effect of attitudes, subjective norms, and perceived behavioral control on behavioral intention were 0.285, 0.366, and 0.322 respectively. The path coefficients for the effect of behavioral intention and perceived behavioral control on prescribing behavior were 0.009 and 0.410 respectively. Conclusion: Physicians' attitudes, subjective norms, and perceived behavioral control were significant positive correlation predictors of behavioral intention. Subjective norms and perceived behavior control had a greater impact than attitude on physicians' prescribing intention. However, the generic prescribing behavior is not under the volitional control of Chinese physicians. Physicians' prescribing practice is likely affected by perceived strong control over prescribing generic drugs.

Incidence, Predictors, and Outcomes of Paravalvular Regurgitation after TAVR in Sievers Type-1 Bicuspid Aortic Valves.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) in patients with bicuspid aortic valve (BAV) stenosis is technically challenging and is burdened by an increased risk of paravalvular regurgitation (PVR). OBJECTIVES: To identify the incidence, predictors, and clinical outcomes of PVR following TAVR in Sievers type 1 BAV stenosis. METHODS: Consecutive patients with severe Sievers type 1 BAV stenosis undergoing TAVR with current generation transcatheter heart valves (THVs) in 24 international centres were enrolled. PVR was graded as none/trace, mild, moderate, and severe according to echocardiographic criteria. The endpoint of major adverse events (MAE), defined as a composite of all-cause death, stroke, or hospitalization for heart failure, was assessed at the last available follow-up. RESULTS: A total of 946 patients were enrolled. PVR occurred in 423 patients (44.7%): mild, moderate, and severe in 387 (40.9%), 32 (3.4%), and 4 (0.4%) patients, respectively. Independent predictors of moderate or severe PVR were larger virtual raphe ring (VRR) perimeter (ORadj 1.07, 95% CI 1.02-1.13), severe annular or left ventricular outflow tract (LVOT) calcification (ORadj 5.21, 95% CI 1.45-18.77), self-expanding valve (ORadj 9.01, 95% CI 2.09-38.86), and intentional supra-annular THV positioning (ORadj 3.31, 95% CI 1.04-10.54). At a median follow-up of 1.3 [IQR 0.5-2.4] years, moderate or severe PVR was associated with an increased risk of MAE (HRadj 2.52, 95% CI 1.24-5.09). CONCLUSIONS: After TAVR with current-generation THVs in Sievers type 1 BAV stenosis, moderate or severe PVR occurred in about 4% of cases and was associated with an increased risk of MAE during follow-up.

A clinical-radiomics nomogram based on spectral CT multi-parameter images for preoperative prediction of lymph node metastasis in colorectal cancer.

To develop a clinical-radiomics nomogram based on spectral CT multi-parameter images for predicting lymph node metastasis in colorectal cancer. A total of 76 patients with colorectal cancer and 156 lymph nodes were included. The clinical data of the patients were collected, including gender, age, tumor location and size, preoperative tumor markers, etc. Three sets of conventional images in the arterial, venous, and delayed phases were obtained, and six sets of spectral images were reconstructed using the arterial phase spectral data, including virtual monoenergetic images (40 keV, 70 keV, 100 keV), iodine density maps, iodine no water maps, and virtual non-contrast images. Radiomics features of lymph nodes were extracted from the above images, respectively. Univariate analysis and least absolute shrinkage and selection operator (LASSO) regression were used to select features. A clinical model was constructed based on age and carcinoembryonic antigen (CEA) levels. The radiomics features selected were used to generate a composed radiomics signature (Com-RS). A nomogram was developed using age, CEA, and the Com-RS. The models' prediction efficiency, calibration, and clinical application value were evaluated by the area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis, respectively. The nomogram outperforms the clinical model and the Com-RS (AUC = 0.879, 0.824). It is well calibrated and has great clinical application value. This study developed a clinical-radiomics nomogram based on spectral CT multi-parameter images, which can be used as an effective tool for preoperative personalized prediction of lymph node metastasis in colorectal cancer.

Congener-specific fate and impact of microcystins in the soil-earthworm system.

Microcystins (MCs) have a significant influence on aquatic ecosystems, but little is known about their terrestrial fate and impact. Here, we investigated the fate of two MCs (MC-LR and MC-RR) in the soil-earthworm system, with consideration of their congener-specific impact on earthworm health, soil bacteria, and soil metabolome. Although MCs had little acute lethal effect on earthworms, they caused obvious growth inhibition and setae rupture. Relative to MC-RR, MC-LR exhibited higher bioaccumulation and the resulting dermal lesions and deformation of longitudinal muscles. While the incorporation of both MCs into soils stimulated pathogenic bacteria and depressed oxidative stress tolerant bacteria, the response among soil nitrification and glutathione metabolism differed between the two congeners. The dissipation kinetics of MCs obeyed the first-order model. Earthworms stimulated soil N-cycling enzyme activities, increased the abundance of MC-degrading bacteria, and promoted bacterial metabolic functions related to glutathione metabolism, xenobiotics biodegradation, and metabolism of amino acids that comprise MCs, which accelerated the dissipation of MC-LR and MC-RR by 227% and 82%, respectively. These results provide evidence of significant congener differences in the terrestrial fate and impact of MCs, which will enable a better understanding of their role in mediating soil functions and ecosystem services.

GABAergic synapses from the ventral lateral septum to the paraventricular nucleus of hypothalamus modulate anxiety.

Emotional disorders, such as anxiety and depression, represent a major societal problem; however, the underlying neurological mechanism remains unknown. The ventral lateral septum (LSv) is implicated in regulating processes related to mood and motivation. In this study, we found that LSv GABAergic neurons were significantly activated in mice experiencing chronic social defeat stress (CSDS) after exposure to a social stressor. We then controlled LSv GABAergic neuron activity using a chemogenetic approach. The results showed that although manipulation of LSv GABAergic neurons had little effect on anxiety-like behavioral performances, the activation of LSv GABAergic neurons during CSDS worsened social anxiety during a social interaction (SI) test. Moreover, LSv GABAergic neurons showed strong projections to the paraventricular nucleus (PVN) of the hypothalamus, which is a central hub for stress reactions. Remarkably, while activation of GABAergic LSv-PVN projections induced social anxiety under basal conditions, activation of this pathway during CSDS alleviated social anxiety during the SI test. On the other hand, the chemogenetic manipulation of LSv GABAergic neurons or LSvGABA-PVN projections had no significant effect on despair-like behavioral performance in the tail suspension test. Overall, LS GABAergic neurons, particularly the LSv GABAergic-PVN circuit, has a regulatory role in pathological anxiety and is thus a potential therapeutic target for the treatment of emotional disorders.

Fungal Plasma Membrane H+-ATPase: Structure, Mechanism, and Drug Discovery.

The fungal plasma membrane H+-ATPase (Pma1) pumps protons out of the cell to maintain the transmembrane electrochemical gradient and membrane potential. As an essential P-type ATPase uniquely found in fungi and plants, Pma1 is an attractive antifungal drug target. Two recent Cryo-EM studies on Pma1 have revealed its hexameric architecture, autoinhibitory and activation mechanisms, and proton transport mechanism. These structures provide new perspectives for the development of antifungal drugs targeting Pma1. In this article, we review the history of Pma1 structure determination, the latest structural insights into Pma1, and drug discoveries targeting Pma1.

COAP-Pd Catalyzed Asymmetric Formal [3+2] Cycloaddition for Optically Active Multistereogenic Spiro Cyclopentane-Indandiones Bearing Cyclic N-Sulfonyl Ketimine Skeletons.

We reported a chiral oxamide-phosphine ligand (COAP-Ph)-Pd-catalyzed asymmetric [3+2] cycloaddition reaction between vinyl cyclopropane compounds derived from 1,3-indanedione and 2-vinylcyclopropane-1,1-dicarboxylates with cyclic sulfonyl 1-azadienes. The corresponding reactions provided a series of enantiomerically active spiro cyclopentane-indandione and cyclopentane structures bearing three consecutive stereogenic centers in good yields with good diastereo- and enantioselectivity. The COAP-Pd complex serves not only to promote generation of chiral π-allyl-palladium intermediates and induce the asymmetry of the reaction, but also depress the background reaction.

Biodegradation of phthalate acid esters and whole-genome analysis of a novel Streptomyces sp. FZ201 isolated from natural habitats.

Di-n-butyl phthalate (DBP) is one of the most extensively used phthalic acid esters (PAEs) and is considered to be an emerging, globally concerning pollutant. The genus Streptomyces holds promise as a degrader of various organic pollutants, but PAE biodegradation mechanisms by Streptomyces species remain unsolved. In this study, a novel PAE-degrading Streptomyces sp. FZ201 isolated from natural habitats efficiently degraded various PAEs. FZ201 had strong resilience against DBP and exhibited immediate degradation, with kinetics adhering to a first-order model. The comprehensive biodegradation of DBP involves de-esterification, ß-oxidation, trans-esterification, and aromatic ring cleavage. FZ201 contains numerous catabolic genes that potentially facilitate PAE biodegradation. The DBP metabolic pathway was reconstructed by genome annotation and intermediate identification. Streptomyces species have an open pangenome with substantial genome expansion events during the evolutionary process, enabling extensive genetic diversity and highly plastic genomes within the Streptomyces genus. FZ201 had a diverse array of highly expressed genes associated with the degradation of PAEs, potentially contributing significantly to its adaptive advantage and efficiency of PAE degradation. Thus, FZ201 is a promising candidate for remediating highly PAE-contaminated environments. These findings enhance our preliminary understanding of the molecular mechanisms employed by Streptomyces for the removal of PAEs.

Genome assembly of autotetraploid Actinidia arguta highlights adaptive evolution and enables dissection of important economic traits.

Actinidia arguta, the most widely distributed Actinidia species and the second cultivated species in the genus, can be distinguished from the currently cultivated Actinidia chinensis on the basis of its small and smooth fruit, rapid softening, and excellent cold tolerance. Adaptive evolution of tetraploid Actinidia species and the genetic basis of their important agronomic traits are still unclear. Here, we generated a chromosome-scale genome assembly of an autotetraploid male A. arguta accession. The genome assembly was 2.77 Gb in length with a contig N50 of 9.97 Mb and was anchored onto 116 pseudo-chromosomes. Resequencing and clustering of 101 geographically representative accessions showed that they could be divided into two geographic groups, Southern and Northern, which first diverged 12.9 million years ago. A. arguta underwent two prominent expansions and one demographic bottleneck from the mid-Pleistocene climate transition to the late Pleistocene. Population genomics studies using paleoclimate data enabled us to discern the evolution of the species' adaptation to different historical environments. Three genes (AaCEL1, AaPME1, and AaDOF1) related to flesh softening were identified by multi-omics analysis, and their ability to accelerate flesh softening was verified through transient expression assays. A set of genes that characteristically regulate sexual dimorphism located on the sex chromosome (Chr3) or autosomal chromosomes showed biased expression during stamen or carpel development. This chromosome-level assembly of the autotetraploid A. arguta genome and the genes related to important agronomic traits will facilitate future functional genomics research and improvement of A. arguta.

Chiral Phosphine Ligands of COAP and SKP Switched Regiodivergent Asymmetric Allylic Alkylation of MBH Adducts.

The palladium-catalyzed highly regioselective asymmetric allylic alkylation of 3'-indolyl-3-oxindole derivatives with Morita-Baylis-Hillman (MBH) carbonates was developed to facilely construct chiral 3,3'-bisindole derivatives under mild reaction conditions. The regioselectivity (α/γ) of MBH carbonates was efficiently switched in the presence of chiral oxalamide phosphine or spiroketal-based diphosphine/Pd(0) complexes as a chiral catalyst. A series of multifunctional 3,3'-bisindole derivatives with all-carbon quaternary stereogenic centers were obtained in high yields with good to excellent enantio-, diastereo-, and regioselectivity. The present process is endowed with some salient features such as broad substrate scope, N-protecting group-free, excellent stereoselectivity, as well as adjustable regioselectivity.

Ginsenoside Rg3: A Review of its Anticancer Mechanisms and Potential Therapeutic Applications.

BACKGROUND: Traditional Chinese Medicine (TCM) has a long history of treating various diseases and is increasingly being recognized as a complementary therapy for cancer. A promising natural compound extracted from the Chinese herb ginseng is ginsenoside Rg3, which has demonstrated significant anticancer effects. It has been tested in a variety of cancers and tumors and has proven to be effective in suppressing cancer. OBJECTIVE: This work covers various aspects of the role of ginsenoside Rg3 in cancer treatment, including its biological functions, key pathways, epigenetics, and potential for combination therapies, all of which have been extensively researched and elucidated. The study aims to provide a reference for future research on ginsenoside Rg3 as an anticancer agent and a support for the potential application of ginsenoside Rg3 in cancer treatment.

Post-surgery financial toxicity and its influencing factors in colorectal cancer care: A cross-sectional study.

PURPOSE: This study aimed to investigate the influence factors of financial toxicity experienced by colorectal cancer patients after surgery. The results will provide deep insights for developing effective intervention strategies to address this common issue of colorectal cancer care. METHODS: In this cross-sectional study, we recruited 213 postoperative patients with colorectal cancer from February 2023 to July 2023 in two major public hospitals. Patients completed the General Information Questionnaire, Comprehensive Scores for Financial Toxicity (COST), Self-perceived Burden Scale (SPBS), Family Resilience Questionnaire (FaREQ), and Social Support Rating Scale (SSRS). A multiple linear regression model was used to investigate the influence factors of financial toxicity. RESULTS: The mean score of financial toxicity was medium (18.91 ± 7.90) in this study. Financial toxicity score was negatively correlated with self-perceived burden (r = -0.333, P < 0.01) and positively associated with family resilience (r = 0.365, P < 0.01) and social support (r = 0.388, P < 0.01). Via multiple linear regression analysis, we identified seven significant factors associated with financial toxicity, including family income [(95 %CI: 1.075-3.123); P = 0.000], self-perceived burden [(95 %CI: 0.300∼-0.038); P = 0.012], stoma [(95 %CI: 5.309∼-1.682); P = 0.000], social support [(95 %CI:0.058-0.407); P = 0.009], cancer stage [(95 %CI: 2.178∼-0.170); P = 0.022], postoperative duration [(95 %CI: 1.900∼-0.332); P = 0.005], and family resilience [(95 %CI: 0.028-0.203); P = 0.010]. CONCLUSIONS: Financial toxicity was prevalent among postoperative colorectal cancer patients. Additional support and early interventions should be given to high-risk patients, including those with stomas, advanced disease stages, or experiencing longer postoperative duration. Apart from demographic factors, we identified that self-perceived burden, family resilience, and social support were also associated with financial toxicity, providing a new perspective for developing effective strategies against financial toxicity.

Safety, immunogenicity and protective effectiveness of heterologous boost with a recombinant COVID-19 vaccine (Sf9 cells) in adult recipients of inactivated vaccines.

Vaccines have proven effective in protecting populations against COVID-19, including the recombinant COVID-19 vaccine (Sf9 cells), the first approved recombinant protein vaccine in China. In this positive-controlled trial with 85 adult participants (Sf9 cells group: n = 44; CoronaVac group: n = 41), we evaluated the safety, immunogenicity, and protective effectiveness of a heterologous boost with the Sf9 cells vaccine in adults who had been vaccinated with the inactivated vaccine, and found a post-booster adverse events rate of 20.45% in the Sf9 cells group and 31.71% in the CoronaVac group (p = 0.279), within 28 days after booster injection. Neither group reported any severe adverse events. Following the Sf9 cells vaccine booster, the geometric mean titer (GMT) of binding antibodies to the receptor-binding domain of prototype SARS-CoV-2 on day 28 post-booster was significantly higher than that induced by the CoronaVac vaccine booster (100,683.37 vs. 9,451.69, p < 0.001). In the Sf9 cells group, GMTs of neutralizing antibodies against pseudo SARS-CoV-2 viruses (prototype and diverse variants of concern [VOCs]) increased by 22.23-75.93 folds from baseline to day 28 post-booster, while the CoronaVac group showed increases of only 3.29-10.70 folds. Similarly, neutralizing antibodies against live SARS-CoV-2 viruses (prototype and diverse VOCs) increased by 68.18-192.67 folds on day 14 post-booster compared with the baseline level, significantly greater than the CoronaVac group (19.67-37.67 folds). A more robust Th1 cellular response was observed with the Sf9 cells booster on day 14 post-booster (mean IFN-γ+ spot-forming cells per 2 × 105 peripheral blood mononuclear cells: 26.66 vs. 13.59). Protective effectiveness against symptomatic COVID-19 was approximately twice as high in the Sf9 cells group compared to the CoronaVac group (68.18% vs. 36.59%, p = 0.004). Our study findings support the high protective effectiveness of heterologous boosting with the recombinant COVID-19 vaccine (Sf9 cells) against symptomatic COVID-19 of diverse SARS-CoV-2 variants of concern, while causing no apparent safety concerns.

5-HT1A receptors within the intermediate lateral septum modulate stress vulnerability in male mice.

Chronic stress is a major risk factor for psychiatric disorders. However, certain individuals may be at higher risk due to greater stress susceptibility. Elucidating the neurobiology of stress resilience and susceptibility may facilitate the development of novel strategies to prevent and treat stress-related disorders such as depression. Mounting evidence suggests that the serotonin (5-HT) system is a major regulator of stress sensitivity. In this study, we assessed the functions of 5-HT1A and 5-HT2A receptors within the lateral septum (LS) in regulating stress vulnerability. Among a group of male mice exposed to chronic social defeat stress (CSDS), 47.2% were classified as stress-susceptible, and these mice employed more passive coping strategies during the defeat and exhibited more severe anxiety- and depression-like behaviors during the following behavioral tests. These stress-susceptible mice also exhibited elevated neuronal activity in the LS as evidenced by greater c-Fos expression, greater activity of 5-HT neurons in both the dorsal and median raphe nucleus, and downregulated expression of the 5-HT1A receptor in the intermediate LS (LSi). Finally, we found the stress-induced social withdrawal symptoms could be rapidly relieved by LSi administration of 8-OH-DPAT, a 5-HT1A receptor agonist. These results indicate that 5-HT1A receptors within the LSi play an important role in stress vulnerability in mice. Therefore, modulation of stress vulnerable via 5-HT1A receptor activation in the LSi is a potential strategy to treat stress-related psychiatric disorders.

Prognosis prediction and risk stratification of breast cancer patients based on a mitochondria-related gene signature.

As the malignancy with the highest global incidence, breast cancer represents a significant threat to women's health. Recent advances have shed light on the importance of mitochondrial function in cancer, particularly in metabolic reprogramming within tumors. Recognizing this, we developed a novel risk signature based on mitochondrial-related genes to improve prognosis prediction and risk stratification in breast cancer patients. In this study, transcriptome data and clinical features of breast cancer samples were extracted from two sources: the TCGA, serving as the training set, and the METABRIC, used as the independent validation set. We developed the signature using LASSO-Cox regression and assessed its prognostic efficacy via ROC curves. Furthermore, the signature was integrated with clinical features to create a Nomogram model, whose accuracy was validated through clinical calibration curves and decision curve analysis. To further elucidate prognostic variations between high and low-risk groups, we conducted functional enrichment and immune infiltration analyses. Additionally, the study encompassed a comparison of mutation landscapes and drug sensitivity, providing a comprehensive understanding of the differing characteristics in these groups. Conclusively, we established a risk signature comprising 8 mitochondrial-related genes-ACSL1, ALDH2, MTHFD2, MRPL13, TP53AIP1, SLC1A1, ME3, and BCL2A1. This signature was identified as an independent risk predictor for breast cancer patient survival, exhibiting a significant high hazard ratio (HR = 3.028, 95%CI 2.038-4.499, P < 0.001). Patients in the low-risk group showed a more favorable prognosis, with enhanced immune infiltration, distinct mutation landscapes, and greater sensitivity to anti-tumor drugs. In contrast, the high-risk group exhibited an adverse trend in these aspects. This risk signature represents a novel and effective prognostic indicator, suggesting valuable insights for patient stratification in breast cancer.

Febrile infection-related epilepsy syndrome with claustrum lesion: an underdiagnosed inflammatory encephalopathy.

OBJECTIVE: To summarize the clinical characteristics and prognosis of febrile infection-related epilepsy syndrome with claustrum lesions (FIRES-C). METHOD: Clinical data of FIRES-C patients were collected retrospectively. The study reviewed and analyzed their clinical manifestations, treatment strategies, and prognosis. RESULT: Twenty patients were enrolled, including 13 females and 7 males, with a median onset age of 20.5 years. All patients developed seizures after fever, with a median interval of 5 days. Brain MRI showed symmetric lesions in the claustrum in all patients. The median interval from seizure onset to abnormal MRI signals detection was 12.5 days. All patients had negative results for comprehensive tests of neurotropic viruses and antineuronal autoantibodies. Seventy percent of cases had been previously empirically diagnosed with autoimmune encephalitis or viral encephalitis before. All patients received anti-seizure medicine. Eleven patients (55%) received antiviral therapy. All patients received immunotherapy, including glucocorticoids (100%), intravenous immunoglobulin (IVIg) (65%), plasma exchange (PLEX) (10%), tocilizumab (10%), rituximab (5%), and cyclophosphamide (5%). Sixty percent of patients received long-term immunotherapy (≥ 3 months). The median follow-up was 11.5 months;60% of patients were diagnosed with refractory epilepsy. CONCLUSION: Bilateral claustrum lesion on MRI is a distinctive neuroimage feature for FIRES, which may serve as an indication for the initial clinical assessments. FIRES-C should be classified as a type of inflammatory encephalopathy characterized by a monophasic nature. Some FIRES-C patients respond to immunotherapy and antiseizure treatments but most experience refractory epilepsy as a long-term outcome.