Levodopa-induced dyskinesia in early-onset Parkinson's disease (EOPD) associates with glucocerebrosidase mutation: A next-generation sequencing study in EOPD patients in Thailand.

BACKGROUND: With the benefit of using next-generation sequencing (NGS), our aim was to examine the prevalence of known monogenic causes in early-onset Parkinson's disease (EOPD) patients in Thailand. The association between clinical features, such as levodopa-induced dyskinesia (LID), and genotypes were also explored. METHOD: NGS studies were carried out for EOPD patients in the Tertiary-referral center for Parkinson's disease and movement disorders. EOPD patients who had LID symptoms were enrolled in this study (n = 47). We defined EOPD as a patient with onset of PD at or below 50 years of age. LID was defined as hyperkinetic movements including chorea, ballism, dystonia, myoclonus, or any combination of these movements resulting from levodopa therapy, which could be peak-dose, off-period, or diphasic dyskinesias. RESULTS: Pathogenic variants were identified in 17% (8/47) of the Thai EOPD patients, of which 10.6% (5/47) were heterozygous GBA variants (c.1448T>C in 3 patients and c.115+1G>A in 2 patients), 4.3% (2/47) homozygous PINK1 variants (c.1474C>T) and 2.1% (1/47) a PRKN mutation (homozygous deletion of exon 7). The LID onset was earlier in patients with GBA mutations compared to those without (34.8±23.4 vs 106.2±59.5 months after starting levodopa, respectively, p = 0.001). LID onset within the first 30 months of the disease was also found to be independently associated with the GBA mutation (odds ratio [95% confidence interval] = 25.00 [2.12-295.06], p = 0.011). CONCLUSION: Our study highlights the high prevalence of GBA pathogenic variants in Thai patients with EOPD and the independent association of these variants with the earlier onset of LID. This emphasizes the importance of genetic testing in this population.

Nine patients with KCNQ2-related neonatal seizures and functional studies of two missense variants.

Mutations in KCNQ2 encoding for voltage-gated K channel subunits underlying the neuronal M-current have been associated with infantile-onset epileptic disorders. The clinical spectrum ranges from self-limited neonatal seizures to epileptic encephalopathy and delayed development. Mutations in KCNQ2 could be either gain- or loss-of-function which require different therapeutic approaches. To better understand genotype-phenotype correlation, more reports of patients and their mutations with elucidated molecular mechanism are needed. We studied 104 patients with infantile-onset pharmacoresistant epilepsy who underwent exome or genome sequencing. Nine patients with neonatal-onset seizures from unrelated families were found to harbor pathogenic or likely pathogenic variants in the KCNQ2 gene. The p.(N258K) was recently reported, and p. (G279D) has never been previously reported. Functional effect of p.(N258K) and p.(G279D) has never been previously studied. The cellular localization study demonstrated that the surface membrane expression of Kv7.2 carrying either variant was decreased. Whole-cell patch-clamp analyses revealed that both variants significantly impaired Kv7.2 M-current amplitude and density, conductance depolarizing shift in voltage dependence of activation, membrane resistance, and membrane time constant (Tau), indicating a loss-of-function in both the homotetrameric and heterotetrameric with Kv7.3 channels. In addition, both variants exerted dominant-negative effects in heterotetrameric with Kv7.3 channels. This study expands the mutational spectrum of KCNQ2- related epilepsy and their functional consequences provide insights into their pathomechanism.

Student academic performance in non-lecture physiology topics following the abrupt change from traditional on-site teaching to online teaching during COVID-19 pandemic.

BACKGROUND: During the COVID-19 pandemic, pre-clerkship medical education, including all physiology classes, was obliged to change to online teaching due to limitations of on-site (face-to-face) classes. However, the effectiveness of online teaching in non-lecture physiology topics during the COVID-19 pandemic has not been thoroughly investigated. METHOD: We conducted a prospective study to evaluate the students' academic achievement and opinions on online teaching during the COVID-19 academic year. Academic achievement of 312 students in the COVID-19 year was compared with that of 299 students in the pre-COVID-19 year. Student opinions regarding social interactions and the preferred learning method were also collected. RESULTS: We found that student academic achievement in the non-lecture physiology topics, assessed by summative scores, was 4.80±0.92 percent higher in the pre-COVID-19 year than in the COVID-19 year (P < 0.01, Cohen's d = 0.42). Students rated that online classes tended to reduce their interactions with peers and teachers; however, students preferred online learning over traditional on-site learning. CONCLUSIONS: This study pointed out that students' academic performance related to the physiology topics taught by online non-lecture methods during the COVID-19 pandemic was lower than their performance when the topics were taught by the traditional (on-site) methods, although students reported that they preferred the online teaching. Hence, we suggest that medical teachers should deliberately plan and utilise a variety of tools and techniques when developing online non-lecture classes to preserve the interactivity of the classes, which might overcome this gap in students' academic performance.

Monoamine Levels and Parkinson's Disease Progression: Evidence From a High-Performance Liquid Chromatography Study.

Parkinson's disease (PD) is associated with dysfunction of monoamine neurotransmitter systems. We investigated changes in the levels of monoamine and their metabolites in PD patients, together with their association to clinical profiles. PD patients and age-matched control subjects (n = 40 per group) were enrolled. Using high-performance liquid chromatography (HPLC) with an electrochemical detector, levels of monoamines (dopamine, DA; norepinephrine, NE; epinephrine, EPI; and serotonin, 5-HT) were measured in plasma, while the metabolites (homovanillic acid, HVA; vanillylmandelic acid, VMA; and 5-hydroxyindoleacetic acid, 5-HIAA) were measured in urine. Plasma DA level was not significantly different between PD and control groups. PD patients had significantly higher plasma NE but lower EPI and 5-HT levels. PD patients had a significantly higher HVA/DA ratio and lower VMA/NE ratio than control subjects, while the 5-HIAA/5-HT ratio was not different between the groups. Regarding the association between monoamine levels and clinical profiles, the DA level had a negative relationship with disease duration and the 5-HT level had a negative relationship with severity of motor impairment. These findings emphasized the involvements of several neurotransmission systems and their association with clinical profiles in PD patients, demonstrated by quantification of monoamine levels in peripheral body fluids. This could benefit appropriate pharmacological treatment planning in respect of monoamine changes and might also help predict subsequent clinical symptoms.

MCM-2, Ki-67, and EGFR downregulated expression levels in advanced stage laryngeal squamous cell carcinoma.

We present the conceptual study investigated the capacity of minichromosome maintenance-2 (MCM-2), Ki-67, and epidermal growth factor receptor (EGFR) to assess the severity and progression of laryngeal squamous cell carcinoma (LSCC) disease and to study the correlations among these markers. A total of 30 patients with LSCC with immunohistochemistry (IHC) staining for MCM-2, Ki-67 and EGFR were examined. Mean expression levels of the three markers were evaluated for comparing between early and advanced stages of LSCC. The mean MCM-2, Ki-67, and EGFR expression levels were significantly decreased in advanced-stage compared with early-stage LSCC. Pearson correlation analysis showed a statistically significant correlation between the MCM-2 and Ki-67. Regarding subgroup analyses, MCM-2, Ki-67, and EGFR showed significant differences between early- and advanced-stage LSCC with non-recurrence, while for the recurrent subgroup LSCC, only MCM-2 revealed a significant difference between early- and advanced-stage LSCC. Altogether, these results support the role for downregulation of MCM-2, Ki-67 and EGFR in advanced-stage LSCC and correlation of MCM-2 and Ki-67 expressions that would be a promising strategy to predict prognosis of LSCC including severity and progression. We contextualize our findings and advocate the position of the biological markers, especially MCM-2, as an emerging evaluation tool for LSCC disease.

The concept-sharing approach: a teaching strategy to promote objective-oriented learning and academic performance in medical students.

Traditionally teachers display the learning outline at the beginning and conclusions at the end of didactic teaching sessions, and students may find it difficult to understand how teaching activities relate to learning objectives and what they should study for assessments. We introduced the "concept-sharing approach" in our neurophysiology course. This approach explicates how the content relates with learning objectives throughout the entirety of each teaching session to help the students assimilate the learning material and direct their learning in an objective-orientated way. In this study, we aimed to examine satisfaction of the concept-sharing approach in medical students and to investigate the relationship between student perception of this teaching strategy and academic performance. The results demonstrated that most participants (74.1%) had a positive impression of this teaching strategy at the end of the course, which was significantly >50.7% at the beginning of the course (P < 0.001). The participants who agreed the concept-sharing approach was useful had significantly higher final assessment (P = 0.014) and end-of-course formative assessment scores (P = 0.001). The findings indicate that after experiencing this new teaching approach, medical students appreciate its utility and that students who engage with this approach are more likely to perform well in assessments. In summary, the concept-sharing approach is a simple teaching strategy which was favored by students and may promote academic performance.

EGFR cut-off point for prognostic impact in laryngeal squamous cell carcinoma.

Background: Various molecular biomarkers, including epidermal growth factor receptor (EGFR), have gained importance as predictors of head and neck cancer disease progression.Aims: This study aimed to investigate the ability of EGFR expression as a prognostic marker for laryngeal squamous cell carcinoma (LSCC).Materials and methods: A total of 31 patients with LSCC with immunohistochemistry (IHC) staining for EGFR were examined. Digital image processing was applied to analyze EGFR staining intensity and percent distribution, which were calculated as the H-score. We used a receiver operating characteristic (ROC) curve to identify the best cut-off point of EGFR expression, with H-score separated into high- and low-grade for cancer recurrence prediction.Results: The cut-off point of EGFR expression for high- and low-grades was an H-score of 170 with a sensitivity of 75% and a specificity of 66.7%. Using this cut-off, 14 (45.16%) and 17 (54.84%) patients were categorized as having high- and low-grades EGFR, respectively. The analysis showed a significant reverse correlation between the EGFR grade and LSCC recurrence (RR, 0.4; 95% CI, 0.17-0.98; p = .02).Conclusions: Our study demonstrated that EGFR grading using H-score with the generated cut-off point by the ROC curve might be further applied as a potential marker for LSCC prognostic prediction.

Attitude, but not self-evaluated knowledge, correlates with academic performance in physiology in Thai medical students.

Positive attitude and self-evaluation are necessary for medical students and doctors. To explore how best to integrate physiology teaching in our Thai medical curriculum, we investigated relationships between student's academic performance, their attitude, self-evaluated knowledge, and proportion of physiology taught in an organ-system integrated block. We organized 13 physiology laboratory classes, during which students self-rated attitude and knowledge. Academic performance was measured by formative and summative assessments. One hundred thirty-six participants were categorized into most proactive (Most PA), more proactive (More PA), less proactive (Less PA), and least proactive (Least PA) attitude groups by self-preparation questionnaire. Eighty participants were categorized into high (HighE), moderate (ModerateE), and low (LowE) self-evaluation rating groups. Mean formatives score in the Most PA group was significantly higher than in the other PA groups (P = 0.003, P = 0.001, and P < 0.001, respectively). Mean summative score in the Most PA group was significantly higher than the Less PA and the Least PA groups (P = 0.017 and P = 0.015 respectively). There was no significant difference in mean assessment scores among HighE, ModerateE, and LowE groups. Proportion of teaching time dedicated to physiology positively correlated with student attitude (r = 0.84, P = 0.001) and negatively correlated with self-evaluation rating (r = -0.73, P = 0.007). Thai medical students may benefit from a proactive attitude to studying physiology, contrasting with traditional didactic expectations of Thai education. Proportion of teaching time dedicated to physiology does not influence academic performance; therefore, future adjustments to curriculum integration may incorporate classes that facilitate self-directed learning. Future study should explore other influences on learning and assessment performance.

Interactive laboratory classes enhance neurophysiological knowledge in Thai medical students.

Interactive laboratory class (ILC) is a two-way communication teaching method that encourages students to correlate laboratory findings with materials from lectures. In Thai medical education, active learning methods are uncommon. This paper aims to establish 1) if ILCs would effectively promote physiology learning; 2) if effectiveness would be found in both previously academically high-performing and low-performing students; and 3) the acceptability of ILCs to Thai medical students as a novel learning method. Two hundred seventy-eight second-year medical students were recruited to this study. We conducted three ILC sessions, which followed corresponding lectures. We carried out multiple-choice pre- and post-ILC assessments of knowledge and compared by repeated-measures ANOVA and unpaired t-test. Subgroup analysis was performed to compare high-performance (HighP) and low-performance (LowP) students. After the ILCs, participants self-rated their knowledge and satisfaction. Post-ILC test scores increased significantly compared with pre-ILC test scores in all three sessions. Mean scores of each post-ILC test increased significantly from pre-ILC test in both LowP and HighP groups. More students self-reported a "very high" and "high" level of knowledge after ILCs. Most students agreed that ILCs provided more discussion opportunity, motivated their learning, and made lessons more enjoyable. As an adjunct to lectures, ILCs can enhance knowledge in medical students, regardless of previous academic performance. Students perceived ILC as useful and acceptable. This study supports the active learning methods in physiology education, regardless of cultural context.

Estrous Cycle Induces Peripheral Sensitization in Trigeminal Ganglion Neurons: An Animal Model of Menstrual Migraine.

BACKGROUND: Many women experience menstrual migraines that develop into recurrent migraine attacks during menstruation. In the human menstrual cycle, the estrogen level fluctuates according to changes in the follicular and luteal phases. The rat estrous cycle is used as an animal model to study the effects of estrogen fluctuation. OBJECTIVE: To investigate whether the estrous cycle is involved in migraine development by comparing the neuronal excitability of trigeminal ganglion (TG) neurons in each stage of the estrous cycle. MATERIAL AND METHOD: Female rats were divided into four experimental groups based on examinations of the cytologies of vaginal smears, and serum analyses of estrogen levels following each stage of the estrous cycle. The rats in each stage of the estrous cycle were anesthetized and their trigeminal ganglia were removed The collections of trigeminal ganglia were cultured for two to three hours, after which whole-cell patch clamp experiments were recorded to estimate the electrophysiological properties of the TG neurons. RESULTS: There were many vaginal epithelial cells and high estrogen levels in the proestrus and estrus stages of the estrous cycle. Electrophysiological studies revealed that the TG neurons in the proestrus and estrus stages exhibited significantly lower thresholds of stimulation, and significant increase in total spikes compared to the TG neurons that were collected in the diestrus stage. CONCLUSION: Our results revealed that high estrogen levels in the proestrus and estrus stages altered the thresholds, rheobases, and total spikes of the TG neurons. High estrogen levels in the estrous cycle induced an increase in neuronal excitability and the peripheral sensitization of TG neurons. These findings may provide an explanation for the correlation of estrogen fluctuations during the menstrual cycle with the pathogenesis of menstrual migraines.

The estrous cycle modulates voltage-gated ion channels in trigeminal ganglion neurons.

Migraines typically occur more frequently in women than men because of the effects of estrogen on both the frequency and severity of migraine attacks. Many women suffer from migraine attacks during menstruation, which are known as menstrual migraines. The pathophysiology of menstrual migraines can be explored by using the rat estrous cycle, which shows a cyclical fluctuation of estrogen level that resembles the menstrual cycle. The aim of this study was to investigate whether different stages of the estrous cycle are involved in migraine development by comparing the excitability of trigeminal ganglion (TG) neurons in four different stages of the estrous cycle by using action potential (AP) parameter assessments. The stages of the estrous cycle were identified by a vaginal smear and measuring the estrogen levels in collected blood. The proestrus and estrus stages had higher estrogen levels compared with the diestrus and metestrus stages. Whole-cell patch clamp recordings demonstrated that TG neurons in the proestrus and estrus stage had lower AP threshold, lower rheobase, higher AP height, shorter AP falling time and deeper afterhyperpolarization (AHP) depth. Hence, our results revealed that the high level of estrogen in the proestrus and estrus stage alters the AP properties of TG neurons. Estrogen may increase membrane excitability and the summation of cellular responses, which alters the AP properties. The alterations of the AP properties in the proestrus and estrus stage may relate to a modification of voltage-gated ion channels in TG neurons, which is a pathogenesis for menstrual migraine. No COI.

GluN2A/B ratio elevation induced by cortical spreading depression: electrophysiological and quantitative studies of the hippocampus.

Cortical spreading depression (CSD), an underlying mechanism of migraine aura, propagates to the hippocampus, and might explain hippocampusassociated symptoms during migraine attack. We hypothesised that this process is, some parts, mediated by NMDA receptors. By using a rat model, CSD was elicited by solid KCl for 45 minutes prior to electrophysiological and quantitative analyses. The result from electrophysiological study was the ratio of glutamate NMDA receptor 2A and 2B subunits (GluN2A/B). Total NMDA receptor response was isolated using an AMPA antagonist, prior to a GluN2B receptor antagonist. The GluN2A/B ratio was calculated by dividing the remaining NMDA-mediated field-excitatory synaptic potentials (fEPSP) with the subtracted difference of NMDAmediated fEPSP. Western blot analysis of the hippocampus was performed to confirm the quantitative change of GluN2A/B ratio. In hippocampal slice study (n = 12), the GluN2A/B ratio of hippocampal fEPSP was significantly increased in CSD group. Western blot analysis (n = 30) revealed an increase in GluN2A subunits and a decrease in GluN2B subunits in the hippocampus ipsilateral to the CSD induction. Our current study revealed that GluN2A/B ratio was shown to be elevated following CSD stimulation by increasing the total number of GluN2A while reducing the total number of GluN2B subunits. This ratio was demonstrated to be associated with synaptic plasticity of the hippocampus in numerous studies. In conclusion, we showed that CSD increased GluN2A/B ratio, in turn, would result in altered synaptic plasticity. Our findings provide a probable implication on the correlation of migraine aura and hippocampusassociated symptoms.

Involvement of AMPA receptors in CSD-induced impairment of LTP in the hippocampus.

OBJECTIVE: To investigate the alteration of hippocampal long-term plasticity and basal synaptic transmission induced by repetitive cortical spreading depressions (CSDs). BACKGROUND: There is a relationship between migraine aura and amnesia attack. CSD, a state underlying migraine attacks, may be responsible for hippocampus-related symptoms. However, the precise role of CSD on hippocampal activity has not been investigated. METHODS: Male Wistar rats were divided into CSD and control groups. Repetitive CSDs were induced in vivo by topical application of solid KCl. Forty-five minutes later, the ipsilateral hippocampus was removed, and hippocampal slices were prepared for a series of electrophysiological studies. RESULTS: Repetitive CSDs led to a decrease in the magnitude of long-term potentiation in the hippocampus. CSD also reduced hippocampal synaptic efficacy, as shown by a reduction in post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses. In contrast, the post-synaptic N-methyl-d-aspartate receptor responses remained unchanged. In addition, there were no changes in paired-pulse profiles between the groups, indicating that CSD did not induce any presynaptic alterations. CONCLUSION: These findings suggest that a reduction of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses is the mechanism responsible for impaired hippocampal long-term potentiation induced by CSD.

Pathophysiology of medication-overuse headache: implications from animal studies.

Recent animal experiments have shown that chronic medication exposure profoundly affects the function of several areas in the nervous system related to headache pathogenesis. These changes include upregulation of calcitonin gene-related peptide, substance P, and nitric oxide synthase in trigeminal ganglia; expansion of receptive field and decreased nociceptive threshold of central trigeminal neurons; decrease in diffuse noxious inhibitory control; and increased susceptibility to develop cortical spreading depression (CSD). These changes indicate an increase in excitability of cortical and trigeminal neurons. The neuronal hyperexcitability may be the result of derangement of a central, possibly serotonin (5-HT)-dependent, modulating control system. Experiments with animals with low 5-HT showed that the processes of CSD and trigeminal nociception are enhanced in this condition. Derangement in the central 5-HT-dependent modulating system as a result of chronic medication use may underlie the chronification of headache as observed in patients with medication-overuse headache.

Nociceptin/orphanin FQ modulates cortical activity and trigeminal nociception.

BACKGROUND: Alterations in the levels of nociceptin/orphanin FQ (N/OFQ) have been reported in patients with primary headaches, including migraines and cluster headaches. These clinical observations suggest that N/OFQ is involved in the pathogenesis of primary headaches. OBJECTIVES: The present study was conducted to determine the role of N/OFQ in the control of trigeminal nociception and cortical excitation. METHODS: Cortical spreading depression (CSD) was elicited in Wistar rats by cortical application of potassium chloride, and electrocorticograms were recorded. N/OFQ was administered via an intracisternal injection. The presence of CSD-evoked trigeminal nociception was determined with Fos and transient receptor potential vanilloid 1 (TRPV1) immunoreactivity. RESULTS: Nociceptin/orphanin FQ produced a biphasic effect on CSD generation, characterized by an initial attenuation followed by delayed potentiation. The amplitude of CSD waves were lower in the initial period but increased in the later period. The total number of CSD waves recorded in 1 hour was greater in the N/OFQ-treated group. Exposure to N/OFQ significantly increased the number of Fos-immunoreactive cells in the trigeminal nucleus caudalis and the number of TRPV1-immunoreactive cells in the trigeminal ganglia, indicating the enhancement of trigeminal nociception. CONCLUSION: These results indicate that N/OFQ can lead to biphasic effect characterized by an initial inhibition, and delay potentiation that eventually intensify CSD-evoked trigeminal nociception.

The neuropeptide nociceptin is a synaptically released endogenous inhibitor of hippocampal long-term potentiation.

Hippocampal long-term potentiation (LTP) of excitatory synaptic transmission has been regarded as a cellular model of learning and memory. Its induction is regulated by many functional molecules at synapses, including the neuropeptide nociceptin, which is identified as an endogenous ligand for the orphan opioid receptor. Mutant mice lacking the receptor exhibit enhanced LTP and hippocampus-dependent memory formation; however, the precise molecular and cellular mechanism is largely unknown. Here, we show that LTP in the hippocampal CA1 region is inhibited by nociceptin synaptically released from interneurons by tetanic stimulation. This endogenous nociceptin downregulates the excitability of pyramidal cells by the hyperpolarization induced by the activation of K(+) channels, which are the common target shared with GABA(B) receptors, although the mode of action is considerably different. Interestingly, the modulation of LTP by endogenous nociceptin is not observed when theta-burst stimulation is used instead of tetanic stimulation, suggesting that relatively longer high-frequency synaptic activation is required for the release of endogenous nociceptin. These results indicate that, in addition to GABA, nociceptin released from interneurons by their high-frequency activation is a novel endogenous neuromodulator that negatively regulates LTP induction in the hippocampus through direct modulation of pyramidal cells.

A biosensing system based on extracellular potential recording of ligand-gated ion channel function overexpressed in insect cells.

We have used outer cell potential measurement to record agonist-dependent cellular responses in cells engineered to express ligand-gated ion channels and grown on a microelectrode surface. Application of glutamate, a natural agonist, induced a complex and robust potentiometric response in cells expressing homomeric GluR-D glutamate receptor, but not in nonexpressing control cells. The response consisted of an initial decrease in outer potential followed by a transient increase and was not obtained for other amino acids devoid of agonist activity at glutamate receptors. Furthermore, the pharmacological agonist of the GluR-D receptor, kainate, also produced the potentiometric response whereas 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive antagonist, was not active in itself but attenuated the responses to glutamate. The time course of the measured changes was slow, which may be partially due to the ligand being applied by free diffusion but may also reflect a contribution by secondary changes in the behavior of the cells. This novel approach should be applicable to other ligand-gated ion channels and holds promise as a cell-based biosensor for high-throughput drug screening and other applications.