Identification of a Novel ERK5 (MAPK7) Inhibitor, MHJ-627, and Verification of Its Potent Anticancer Efficacy in Cervical Cancer HeLa Cells.

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase (MAPK) family, is involved in key cellular processes. However, overexpression and upregulation of ERK5 have been reported in various cancers, and ERK5 is associated with almost every biological characteristic of cancer cells. Accordingly, ERK5 has become a novel target for the development of anticancer drugs as inhibition of ERK5 shows suppressive effects of the deleterious properties of cancer cells. Herein, we report the synthesis and identification of a novel ERK5 inhibitor, MHJ-627, and verify its potent anticancer efficacy in a yeast model and the cervical cancer HeLa cell line. MHJ-627 successfully inhibited the kinase activity of ERK5 (IC50: 0.91 µM) and promoted the mRNA expression of tumor suppressors and anti-metastatic genes. Moreover, we observed significant cancer cell death, accompanied by a reduction in mRNA levels of the cell proliferation marker, proliferating cell nuclear antigen (PCNA), following ERK5 inhibition due to MHJ-627 treatment. We expect this finding to serve as a lead compound for further identification of inhibitors for ERK5-directed novel approaches for oncotherapy with increased specificity.

Premature Mortality and Causes of Death Among People With Epilepsy: A Nationwide Population-Based Incident Cohort Study.

BACKGROUND AND OBJECTIVES: People with epilepsy (PWE) are at risk of premature death with considerable variability according to the study population. We aimed to estimate the risk and causes of death in PWE according to age, disease severity, disease course, comorbidities, and socioeconomic status in Korea. METHODS: We conducted a nationwide population-based retrospective cohort study using the National Health Insurance database linked with the national death register. Newly treated PWE from 2008 to 2016 who were identified by antiseizure medication (ASM) prescriptions and diagnostic codes for epilepsy/seizure were included and observed until 2017. We assessed all-cause and cause-specific crude mortality rates and standardized mortality ratios (SMRs). RESULTS: Among 138,998 PWE, 20,095 deaths were identified, and the mean follow-up period was 4.79 years. The SMR was 2.25 in the overall group of PWE, with a higher value in the younger age group at diagnosis and a shorter time interval after diagnosis. The SMR in the monotherapy group was 1.56, while that in the group with 4 or more ASMs was 4.93. PWE without any comorbidities had an SMR of 1.61. PWE who were rural residents had a higher SMR than those who were urban residents (2.47 vs 2.03, respectively). The causes of death among PWE were cerebrovascular disease (18.9%, SMR 4.50), malignant neoplasms outside the CNS (15.7%, SMR 1.37), malignant neoplasms of the CNS (6.7%, SMR 46.95), pneumonia (6.0%, SMR 2.08), and external causes (7.2%, SMR 2.17), including suicide (2.6%, SMR 2.07). Epilepsy itself and status epilepticus accounted for 1.9% of the overall death. The excess mortality associated with pneumonia and external causes was persistently high, whereas the excess mortality associated with malignancy and cerebrovascular diseases tended to decrease with increasing time since diagnosis. DISCUSSION: This study showed excess mortality in PWE, even in those without comorbidities and those receiving monotherapy. Regional disparities and sustained risks of deaths from external causes over 10 years imply potential points of intervention. In addition to active control of seizures, education about injury prevention, monitoring for suicidal ideation, and efforts to improve accessibility to epilepsy care are all required to reduce mortality.

Synthesis of 1,4-Dialkoxynaphthalene-Based Imidazolium Salts and Their Cytotoxicity in Cancer Cell Lines.

In this study, we designed and synthesized novel 1,4-dialkoxynaphthalene-2-alkyl imidazolium salt (IMS) derivatives containing both 1,4-dialkoxynaphthalene and imidazole, which are well known as pharmacophores. The cytotoxicities of these newly synthesized IMS derivatives were investigated in order to explore the possibility of using them to develop anticancer drugs. It was found that some of the new IMS derivatives showed good cytotoxic activities. In addition, an initial, qualitative structure-activity relationship is presented on the basis of observations of activity changes corresponding to structural changes.

Δ8(14)-Ergostenol Glycoside Derivatives Inhibit the Expression of Inflammatory Mediators and Matrix Metalloproteinase.

Arthritis is a chronic inflammatory disease accompanied by pathological reactions such as swelling, redness, fever, and pain in various joint areas. The drugs currently available to treat arthritis are associated with diverse side-effects. Therefore, there is a need for safer and more effective treatments to alleviate the inflammation of arthritis with fewer side-effects. In this study, a new sterol, Δ8(14)-ergostenol, was discovered, and its glycosides were synthesized and found to be more efficient in terms of synthesis or anti-inflammatory activity than either spinasterol or 5,6-dihydroergosterol is. Among these synthetic glycosides, galactosyl ergostenol inhibited the expression of inflammatory mediators in TNF-α-stimulated FLS and TNF-α-induced MMPs and collagen type II A1 degradation in human chondrocytes. These results suggest the new galactosyl ergostenol as a treatment candidate for arthritis.

The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols.

Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying σ antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.

A case of NMDAR encephalitis treated in the third trimester - novel arterial spin labeling findings and a review of literature.

Diagnosis and treatment of anti-NMDAR encephalitis during pregnancy are challenging. We report a case of anti-NMDAR encephalitis in a 28-year-old, 24 weeks pregnant woman. Her brain magnetic resonance imaging was normal, but arterial spin labeling (ASL) showed increased cerebral blood flow in the right insula and temporal area. She was successfully treated with emergent cesarean section and immunotherapies including intravenous immunoglobulin, steroids, and rituximab. The newborn was healthy and she recovered fully. Early suspicion and proper management is important in treatment of anti-NMDAR encephalitis during pregnancy. ASL can help diagnosing anti-NMDAR encephalitis during pregnancy.

Development of the clinical assessment scale in autoimmune encephalitis.

OBJECTIVE: There is no scale for rating the severity of autoimmune encephalitis (AE). In this study, we aimed to develop a novel scale for rating severity in patients with diverse AE syndromes and to verify the reliability and validity of the developed scale. METHODS: The key items were generated by a panel of experts and selected according to content validity ratios. The developed scale was initially applied to 50 patients with AE (development cohort) to evaluate its acceptability, reproducibility, internal consistency, and construct validity. Then, the scale was applied to another independent cohort (validation cohort, n = 38). RESULTS: A new scale consisting of 9 items (seizure, memory dysfunction, psychiatric symptoms, consciousness, language problems, dyskinesia/dystonia, gait instability and ataxia, brainstem dysfunction, and weakness) was developed. Each item was assigned a value of up to 3 points. The total score could therefore range from 0 to 27. We named the scale the Clinical Assessment Scale in Autoimmune Encephalitis (CASE). The new scale showed excellent interobserver (intraclass correlation coefficient [ICC] = 0.97) and intraobserver (ICC = 0.96) reliability for total scores, was highly correlated with modified Rankin scale (r = 0.86, p < 0.001), and had acceptable internal consistency (Cronbach α = 0.88). Additionally, in the validation cohort, the scale showed high interobserver reliability (ICC = 0.99) and internal consistency (Cronbach α = 0.92). INTERPRETATION: CASE is a novel clinical scale for AE with a high level of clinimetric properties. It would be suitable for application in clinical practice and might help overcome the limitations of current outcome scales for AE. ANN NEUROL 2019;85:352-358.

Surgical Prognostic Value of Epileptic Aura Based on History and Electrical Stimulation.

BACKGROUND AND PURPOSE: We attempted to evaluate the surgical prognostic value of various types of aura in conjunction with the results of other presurgical evaluations in patients with an intracranial ictal onset zone confirmed by invasive studies and focal resection. We also attempted to determine how often the habitual auras could be elicited and to demonstrate the prognostic value of these stimulation-induced auras (SIAs). METHODS: We reviewed retrospectively the records of patients who had undergone intracranial electroencephalography evaluation and focal resective surgery for intractable partial epilepsy between 1995 and 2009. We identified the localizing value and prognostic value of the patients' auras. We correlated the resection of the area with SIA and surgical outcome. RESULTS: Aura was reported in 225 out of 300 patients. Patients with medial temporal lobe epilepsy (TLE) or occipital lobe epilepsy had a higher chance of having aura. The presence of aura, medial TLE, hippocampal sclerosis on pathology, focal lesions on magnetic resonance imaging (MRI), and ipsilateral abnormality on fluorodeoxyglucose-positron emission tomography were significantly correlated with seizure-free outcome. The presence of auditory aura, parietal lobe epilepsy, multifocal epilepsy, and dual pathology was associated with poor outcomes. Multivariate analysis revealed that auditory aura, multifocal epilepsy, hippocampal sclerosis, and lesion on MRI were prognostic factors for intractable partial epilepsy. SIA was observed in 29 out of the 134 patients who had habitual aura on history. The degree of complete resection of the area with SIA was not related to seizure-free outcome. CONCLUSIONS: The presence of aura favors good surgical outcome and certain types of aura, such as auditory aura, have poor prognostic value. SIA, which was encountered in 21.6% of patients, was not related to seizure-free outcome.

Surgical Treatment of Nonlesional Neocortical Epilepsy: Long-term Longitudinal Study.

Importance: The proportion of surgery for nonlesional neocortical epilepsy has recently increased, with a decrease in surgery for mesial temporal lobe epilepsy. However, there are only a few studies regarding the long-term surgical outcome and the potential prognostic factors for patients with nonlesional neocortical epilepsy. Objective: To evaluate the long-term surgical outcome and to identify possible prognostic factors in patients with nonlesional neocortical epilepsy. Design, Setting, and Participants: In a surgical cohort from September 1995 to December 2005 at the Seoul National University Hospital, we included 109 consecutive patients without lesions identifiable by magnetic resonance imaging who underwent focal surgical resection for drug-resistant neocortical epilepsy. Follow-up information for at least 10 years was available for all but 1 patient. Main Outcomes and Measures: Univariate and standard multiple logistic regression analyses were performed to identify the predictors of surgical outcomes, and a generalized estimation equation model was used for the longitudinal multiple logistic regression analysis of up to 21 years of follow-up. Results: The patients consisted of 64 men and 45 women with ages at surgery ranging from 7 to 56 years (mean [SD], 27.1 [7.8] years). At 1 year after surgery, 59 of 109 patients (54.1%) achieved seizure freedom, and 64 of 108 patients (59.3%) achieved seizure freedom at the last follow-up. Only 11 of 108 patients (10.2%) experienced definite changes in postoperative seizure status. Localizing patterns in functional neuroimaging (strongest odds ratio [OR], 0.30 [95% CI, 0.14-0.66] for fluorodeoxyglucose-positron emission tomography; 0.37 [95% CI, 0.15-0.87] for ictal single-photon emission computed tomography), concordant results in presurgical diagnostic evaluations (OR, 3.15 [95% CI, 1.42-7.02]), the presence of aura (OR, 3.49 [95% CI, 1.54-7.92]), and complete resection of areas of ictal onset with frequent interictal spikes during the intracranial electroencephalographic study (OR, 0.37 [95% CI, 0.16-0.85]) were favorable surgical outcome predictors. Conclusions and Relevance: Our study showed that nearly 60% of patients with nonlesional neocortical epilepsy achieved freedom from long-term seizure, and that changes in postoperative seizure status were rarely observed. Several predictors of favorable surgical outcomes were identified, which can help select optimal candidates for surgical treatment among patients with nonlesional neocortical epilepsy.

Change of Patient Selection Strategy and Improved Surgical Outcome in MRI-negative Neocortical Epilepsy.

BACKGROUND AND PURPOSE: It is crucial to make selection strategy to identify surgical candidates among medically refractory MRI-negative neocortical epilepsy patients. In our previous study, we suggested two or more concordance between noninvasive studies (EEG, ictal scalp EEG, interictal FDG-PET, and SPECT) as a new patient selection strategy for MRI-negative neocortical epilepsy surgery. The objective of this study was to evaluate the surgical outcomes of MRI-negative neocortical epilepsy patients before and after the implementation of a new selection strategy. METHODS: From 1995 to 2011, we included 153 consecutive MRI-negative neocortical epilepsy patients who received focal resection and had a follow-up period of at least 2 years. These patients were divided into two groups according to their date of surgery (before and after July 2002). The old group consisted of 89 patients and the new one consisted of 53 patients. Clinical characteristics, presurgical evaluations, and pathology were reviewed. RESULTS: The new patient selection strategy led to a significant increase in the concordance between two or more modalities. The improvement in surgical outcome after 2002 was significant (seizure-free outcome, 47.2% vs. 75.5%; p = 0.001). Concordance between two or more presurgical evaluations and localizing PET were related to a seizure-free outcome in a multivariate analysis. CONCLUSIONS: After a change in surgical strategy to select patients with two or more concordance between noninvasive studies, the seizure-free outcome improved up to 75.5%. MRI-negative neocortical epilepsy patients with two or more concordance between noninvasive studies seem to be good candidates for epilepsy surgery.

A comparison of MRI tissue relaxometry and ROI methods used to determine regional brain iron concentrations in restless legs syndrome.

PURPOSE: Magnetic resonance imaging relaxometry studies differed on the relaxometry methods and their approaches to determining the regions of interest (ROIs) in restless legs syndrome (RLS) patients. These differences could account for the variable and inconsistent results found across these studies. The aim of this study was to assess the relationship between the different relaxometry methods and different ROI approaches using each of these methods on a single population of controls and RLS subjects. METHODS: A 3.0-T magnetic resonance imaging with the gradient-echo sampling of free induction decay and echo pulse sequence was used. The regional brain "iron concentrations" were determined using three relaxometry metrics (R2, R2*, and R2') through two different ROI methods. The substantia nigra (SN) was the primary ROI with red nucleus, caudate, putamen, and globus pallidus as the secondary ROIs. RESULTS: Thirty-seven RLS patients and 40 controls were enrolled. The iron concentration as determined by R2 did not correlate with either of the other two methods, while R2* and R2' showed strong correlations, particularly for the substantia nigra and red nucleus. In the fixed-shape ROI method, the RLS group showed a lower iron index compared to the control group in the substantia nigra and several other regions. With the semi-automated ROI method, however, only the red nucleus showed a significant difference between the two groups. CONCLUSION: Both the relaxometry and ROI determination methods significantly influenced the outcome of studies that used these methods to estimate regional brain iron concentrations.

On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device.

For tissue engineering and regenerative medicine, cryopreservation, a technique for preserving biomaterials in the frozen state with cryoprotective agents (CPAs), is critically important for preserving engineered tissues (ETs) as well as cells necessary to create ETs. As more diverse ETs are produced using various cell types, CPAs and corresponding freeze/thaw (F/T) protocols need to be developed cell/tissue-type specifically. This is because CPAs and F/T protocols that have been successful for one cell/tissue type have proven to be difficult to adapt to other cell/tissue types. The most critical barrier to address this challenge is the inability to screen and identify CPA or CPA mixtures efficiently. In this paper, we developed an "electro-wetting-on-dielectic" (EWOD) based digital microfluidic platform to characterize and screen CPA mixtures cell-type specifically. The feasibility of the EWOD platform was demonstrated by characterizing and optimizing a mixture of dimethlysulfoxide (DMSO) and PBS for human breast cancer cell line as model CPA mixture and cell line. The developed platform multiplexed droplets of DMSO and PBS to create an array of DMSO-PBS mixtures, and mapped the phase change diagram of the mixture. After loading cell suspensions on the platform, the mixture was further screened on-chip for toxicity and cryoprotection. The results were discussed to illustrate the capabilities and limitations of the EWOD platform for cell and tissue-type specific optimization of CPA mixtures and F/T protocols.

Electrowetting-based microfluidics for analysis of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry.

A new technique for preparing samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The technique relies on electrowetting-on-dielectric (EWOD) to move droplets containing proteins or peptides and matrix to specific locations on an array of electrodes for analysis. Standard MALDI-MS reagents, analytes, concentrations, and recipes are demonstrated to be compatible with the technique. Mass spectra are comparable to those collected by conventional methods. Nonspecific adsorption of analytes to device surfaces is demonstrated to be negligible. The results suggest that EWOD may be a useful tool for automating sample preparation for high-throughput proteomics and other applications of MALDI-MS.