Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Silver-Russell Syndrome.

Background: Silver-Russell syndrome (SRS; OMIM #180860) is a clinically and genetically heterogeneous imprinting disorder characterized by prenatal and postnatal growth failure. The aim of this study was to identify the epigenotype-phenotype correlations in these patients using quantitative DNA methylation analysis. Methods: One hundred and eighty-three subjects clinically suspected of having SRS were referred for diagnostic testing by the methylation profiling of H19-associated imprinting center (IC) 1 and imprinted PEG1/MEST regions using methylation-specific high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between quantitative DNA methylation status and clinical manifestations of the subjects according to the Netchine-Harbison (N-H) clinical scoring system for SRS were analyzed. Results: Among the 183 subjects, 90 had a clinical diagnosis of SRS [N-H score ≥ 4 (maximum = 6)] and 93 had an SRS score < 4. Molecular lesions were detected in 41% (37/90) of the subjects with a clinical diagnosis of SRS, compared with 3% (3/93) of those with an N-H score < 4. The IC1 methylation level was negatively correlated with the N-H score. The molecular diagnosis rate was positively correlated with the N-H score. Thirty-one subjects had IC1 hypomethylation (IC1 methylation level <35% by the MassARRAY assay), seven had maternal uniparental disomy 7, and two had pathogenic copy number variants. Among the 90 subjects with an N-H score ≥ 4, the IC1 methylation level was significantly different between those with or without some clinical SRS features, including birth length ≤ 10th centile, relative macrocephaly at birth, normal cognitive development, body asymmetry, clinodactyly of the fifth finger, and genital abnormalities. Conclusions: This study confirmed the suitability of the N-H clinical scoring system as clinical diagnostic criteria for SRS. Quantitative DNA methylation analysis using the MassARRAY assay can improve the detection of epigenotype-phenotype correlations, further promoting better genetic counseling and multidisciplinary management for these patients.

Using deep learning to accurately detect sow vulva size in a group pen with a single camera.

This paper presents a non-contact method for the detection of changes in sow vulva size in a group pen. The traditional approach to estrus detection is manually pressing down on the back of the sow to elicit standing responses; however, this method causes undue distress for sows not in estrus. When a sow is in estrus, the vulva is red and swollen due to the presence of endocrine. Monitoring changes in vulva size to detect estrus with as little impact on the sow as possible is the focus of this study. This is achieved using a single camera combined with a deep learning framework. Our approach comprises two steps: vulva detection and vulva size conversion. Images of sows of Yorkshire, Landrace, and Duroc breeds were collected in group housing, and the vulva was detected through artificial markers and the network architecture of YOLO v4. Based on the internal and external parameters of the camera, the detected size was converted into millimeters and the results of manual measurement (MM) and automatic calculation combined to calculate the size of the vulva. Analysis of the calculated size compared with MM indicates that the object recognition rate of the system exceeds 97.06%, with a size error of only + 1.70 to -4.47 mm and high-calculation efficiency (>2.8 frames/s). Directions for future research include the automatic detection of pig width.

The size of a sow's vulva is an important indicator of sow estrus. Non-contact means of monitoring size changes for estrus timing would represent a significant contribution to the field of pig farming. This paper thus focuses on development of a system for the automatic detection of sow vulva size using a single camera combined with a deep learning framework. Experiments showed that the object recognition rate of the system exceeds 97.06%, the vulva size error is +1.70 to −4.47 mm, and the calculation efficiency is high (>2.8 frames/s).

Incidental finding of severe hyperkalemia in a patient with end-stage renal disease during video-assisted lung lobectomy: A case report.

Patients with end-stage renal disease are at risk of developing hyperkalemia and acidosis, both of which have disastrous sequelae during elective video-assisted thoracic surgery for lung cancer. Herein, we present a case where severe hyperkalemia and combined acidosis were incidentally found in a 68-year-old man with the end-stage renal disease after establishing one-lung ventilation during video-assisted lobectomy. There was no significant instability of vital signs, abnormality of perioperative electrocardiography, or malignant arrhythmia. Therefore, we arranged for related management promptly, and the surgery was relatively smooth. This incidental intraoperative hyperkalemia was thought to have resulted from one-lung ventilation and hypercarbia and/or metabolic acidosis. More frequent arterial blood gas analysis and aggressive blood potassium control during video-assisted thoracic surgery should be considered for patients with end-stage renal disease.

Using deep learning to identify maturity and 3D distance in pineapple fields.

Pineapples are an important agricultural economic crop in Taiwan. Considerable human resources are required to protect pineapples from excessive solar radiation, which could otherwise lead to overheating and subsequent deterioration. Note that simple covering all of the fruit with a paper bag is not a viable solution, due to the fact that it makes it impossible to determine whether the fruit is ripe. This paper proposes a system by which to automate the detection of ripe pineapples. The proposed deep learning architecture enables detection regardless of lighting conditions, achieving accuracy of more than 99.27% with error of less than 2% at distances of 300 ~ 800 mm. This proposed system using an Nvidia TX2 is capable of 15 frames per second, thereby making it possible to mount the device on machines that move at walking speed.

Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Beckwith-Wiedemann Syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS; OMIM 130650) is a rare overgrowth syndrome with tumor predisposition resulting from the abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. The aim of this study was to identify the epigenotype-phenotype correlations of these patients using quantitative DNA methylation analysis. METHODS: One hundred and four subjects with clinically suspected BWS were enrolled in this study. All of the subjects had been referred for diagnostic testing which was conducted using methylation profiling of H19-associated imprinting center (IC) 1 and KCNQ1OT1-associated IC2 in high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between the quantitative DNA methylation status and clinical manifestations of the enrolled subjects were analyzed. RESULTS: Among the 104 subjects, 19 had IC2 hypomethylation, 2 had IC1 hypermethylation, and 10 had paternal uniparental disomy (pUPD). The subjects with IC2 hypomethylation were characterized by significantly more macroglossia but less hemihypertrophy compared to the subjects with pUPD (p < 0.05). For 19 subjects with IC2 hypomethylation, the IC2 methylation level was significantly different (p < 0.05) between the subjects with and without features including macroglossia (IC2 methylation level: 11.1% vs. 30.0%) and prenatal or postnatal overgrowth (8.5% vs. 16.9%). The IC2 methylation level was negatively correlated with birth weight z score (p < 0.01, n = 19) and birth height z score (p < 0.05, n = 13). For 36 subjects with clinically diagnosed BWS, the IC2 methylation level was negatively correlated with the BWS score (r = -0.592, p < 0.01). The IC1 methylation level showed the tendency of positive correlation with the BWS score without statistical significance (r = 0.137, p > 0.05). CONCLUSIONS: Lower IC2 methylation and higher IC1 methylation levels were associated with greater disease severity in the subjects with clinically diagnosed BWS. Quantitative DNA methylation analysis using the MassARRAY assay could improve the detection of epigenotype-phenotype correlations, which could further promote better genetic counseling and medical care for these patients.

Epigenotype, Genotype, and Phenotype Analysis of Taiwanese Patients with Silver-Russell Syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous disorder characterized by severe intrauterine growth retardation, poor postnatal growth, characteristic facial features, and body asymmetry. Hypomethylation of the imprinted genes of the chromosome 11p15.5 imprinting gene cluster and maternal uniparental disomy of chromosome 7 (mUPD7) are the major epigenetic disturbances. The aim of this study was to characterize the epigenotype, genotype, and phenotype of these patients in Taiwan. METHODS: Two hundred and six subjects with clinically suspected SRS were referred for diagnostic testing, which was performed by profiling the methylation of H19-associated imprinting center (IC) 1 and the imprinted PEG1/MEST region using methylation-specific multiplex ligation-dependent probe amplification and high-resolution melting analysis with a methylation-specific polymerase chain reaction assay. We also applied a whole genome strategy to detect copy number changes and loss of heterozygosity. Clinical manifestations were recorded and analyzed according to the SRS scoring system proposed by Bartholdi et al. Results: Among the 206 referred subjects, 100 were classified as having a clinical diagnosis of SRS (score ≥ 8, maximum = 15) and 106 had an SRS score ≤ 7. Molecular lesions were detected in 45% (45/100) of the subjects with a clinical diagnosis of SRS, compared to 5% (5/106) of those with an SRS score ≤ 7. Thirty-seven subjects had IC1 hypomethylation, ten subjects had mUPD7, and three subjects had microdeletions. Several clinical features were found to be statistically different (p < 0.05) between the "IC1 hypomethylation" and "mUPD7" groups, including relative macrocephaly at birth (89% vs. 50%), triangular shaped face (89% vs. 50%), clinodactyly of the fifth finger (68% vs. 20%), and SRS score (11.4 ± 2.2 vs. 8.3 ± 2.5). CONCLUSIONS: The SRS score was positively correlated with the molecular diagnosis rate (p < 0.001). The SRS subjects with mUPD7 seemed to have fewer typical features and lower SRS scores than those with IC1 hypomethylation. Careful clinical observation and timely molecular confirmation are important to allow for an early diagnosis and multidisciplinary management of these patients.

Dual-LC PSHG microscopy for imaging collagen type I and type II gels with pixel-resolution analysis.

Collagen of type I (Col I) and type II (Col II) are critical for cartilage and connective tissues in the human body, and several diseases may alter their properties. Assessing the identification and quantification of fibrillar collagen without biomarkers is a challenge. Advancements in non-invasive polarization-resolved second-harmonic generation (PSHG) microscopy have provided a method for the non-destructive investigation of collagen molecular level properties. Here we explored an alternative polarization modulated approach, dual-LC PSHG, that is based on two liquid crystal devices (Liquid crystal polarization rotators, LPRs) operating simultaneously with a laser scanning SHG microscope. We demonstrated that this more accessible technology allows the quick and accurate generation of any desired linear and circular polarization state without any mechanical parts. This study demonstrates that this method can aid in improving the ability to quantify the characteristics of both types of collagen, including pitch angle, anisotropy, and circular dichroism analysis. Using this approach, we estimated the effective pitch angle for Col I and Col II to be 49.7° and 51.6°, respectively. The effective peptide pitch angle for Col II gel was first estimated and is similar to the value obtained for Col I gel in the previous studies. Additionally, the difference of the anisotropy parameter of both collagen type gels was assessed to be 0.293, which reflects the different type molecular fibril assembly. Further, our work suggests a potential method for monitoring and differentiating different collagen types in biological tissues, especially cartilage or connective tissue.

Synthesis and cytotoxic analysis of thiolated xylose derivatives decorated on gold nanoparticles.

The rapid development of metal nanoparticles capped by an organic monolayer offers the possibility to create a whole new variety of products with novel characteristic, functions and applications. Among these, nanoparticles covered with carbohydrates (glyconanoparticles) constitute a good bio-mimetic model of carbohydrate presentation at the cell surface and are currently centered on many glycobiological and biomedical applications. In this study, a series of novel D-xylose gold nanoparticles (AuNPs) with linkages of alkyl or polyethylene glycol have been synthesized via D-xylosethiols, forming self-assembled monolayers on gold nanoparticles. The nano-gold solution, two carbohydrate derivatives and modified nano-gold solution were tested for cytotoxicity to check the biocompatibility. The MTT assay on NIH 3T3 cell lines confirmed that all the test materials showed no toxicity with the more than 90 % of cell viability in both low concentration (1 µM) and high concentration (100 µM), compared with the control.

Survival and diagnostic age of 175 Taiwanese patients with mucopolysaccharidoses (1985-2019).

BACKGROUND: Mucopolysaccharidoses (MPSs) are a group of inherited metabolic diseases, which are characterized by the accumulation of glycosaminoglycans, and eventually lead to the progressive damage of various tissues and organs. METHODS: An epidemiological study of MPS in Taiwan was performed using multiple sources. The survival and diagnostic age for different types of MPS between 1985 and 2019 were evaluated. RESULTS: Between 1985 and 2019, there were 175 patients diagnosed with MPS disorders in the Taiwanese population, with a median diagnostic age of 3.9 years. There were 21 (12%), 78 (45%), 33 (19%), 32 (18%) and 11 (6%) patients diagnosed with MPS I, II, III, IV and VI, respectively, with median diagnostic ages of 1.5, 3.8, 4.7, 4.5 and 3.7 years, respectively. Diagnosis of MPS patients was significantly earlier in recent decades (p < 0.01). Pilot newborn screening programs for MPS I, II, VI, IVA, and IIIB were progressively introduced in Taiwan from 2016, and 48% (16/33) of MPS patients diagnosed between 2016 and 2019 were diagnosed by one of these screening programs, with a median diagnostic age at 0.2 years. For patients born between 2016 and 2019, up to 94% (16/17) were diagnosed with MPS via the newborn screening programs. At the time of this study, 81 patients had passed away with a median age at death of 15.6 years. Age at diagnosis was positively correlated with life expectancy (p < 0.01). Life expectancy also significantly increased between 1985 and 2019, however this increase was gradual (p < 0.01). CONCLUSIONS: The life expectancy of Taiwanese patients with MPS has improved in recent decades and patients are being diagnosed earlier. Because of the progressive nature of the disease, early diagnosis by newborn screening programs and timely implementation of early therapeutic interventions may lead to better clinical outcomes.

An At-Risk Population Screening Program for Mucopolysaccharidoses by Measuring Urinary Glycosaminoglycans in Taiwan.

BACKGROUND: The mucopolysaccharidoses (MPSs) are a group of rare lysosomal storage disorders characterized by the accumulation of glycosaminoglycans (GAGs) and which eventually cause progressive damage to various tissues and organs. We developed a feasible MPS screening algorithm and established a cross-specialty collaboration platform between medical geneticists and other medical specialists based on at-risk criteria to allow for an earlier confirmative diagnosis of MPS. METHODS: Children (<19 years of age) with clinical signs and symptoms compatible with MPS were prospectively enrolled from pediatric clinics between July 2013 and June 2018. Urine samples were collected for a non-specific total GAG analysis using the dimethylmethylene blue (DMB) spectrophotometric method, and the quantitation of three urinary GAGs (dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)) was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The subjects with elevated urinary GAG levels were recalled for leukocyte enzyme activity assay and genetic testing for confirmation. RESULTS: Among 153 subjects enrolled in this study, 13 had a confirmative diagnosis of MPS (age range, 0.6 to 10.9 years-three with MPS I, four with MPS II, five with MPS IIIB, and one with MPS IVA). The major signs and symptoms with regards to different systems recorded by pediatricians at the time of the decision to test for MPS were the musculoskeletal system (55%), followed by the neurological system (45%) and coarse facial features (39%). For these 13 patients, the median age at the diagnosis of MPS was 2.9 years. The false negative rate of urinary DMB ratio using the dye-based method for these 13 patients was 31%, including one MPS I, two MPS IIIB, and one MPS IVA. However, there were no false negative results with urinary DS, HS and KS using the MS/MS-based method. CONCLUSIONS: We established an at-risk population screening program for MPS by measuring urinary GAG fractionation biomarkers using the LC-MS/MS method. The program included medical geneticists and other medical specialists to increase awareness and enable an early diagnosis by detecting MPS at the initial onset of clinical symptoms.

Functional independence of Taiwanese patients with mucopolysaccharidoses.

BACKGROUND: Information on functional strengths and weaknesses of mucopolysaccharidosis (MPS) patients is important for early intervention programs and enzyme replacement therapy (ERT). METHODS: We used the Functional Independence Measure for Children (WeeFIM) questionnaire to assess the functional skills of 63 Taiwanese MPS patients (median age, 13 years 3 months; range, 3-20 years) from January 2012 to December 2018. RESULTS: Mean total WeeFIM score was 75.4 of a potential score of 126. Mean total WeeFIM scores of each type (MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI) were 103.8, 76.2, 41.6, 92.2, and 113.6, respectively. Mean scores for self-care, mobility, and cognition domains were 30 (maximum 56), 23 (maximum 35), and 22 (maximum 35), respectively. MPS type IIIB patients had the lowest scores in self-care, mobility, cognition, and total domains compared to other types of MPS. All patients with ERT in MPS I, II, and IVA had higher scores in self-care and mobility domains than patients without ERT. Most patients required assistance for self-care skills, especially in grooming and bathing. CONCLUSION: MPS patients require support and supervision in self-care tasks. For cognition tasks, MPS IIIB patients also require help. This questionnaire is useful to identify the strengths and limitations of MPS patients.

Ciproxifan, a histamine H3 receptor antagonist and inverse agonist, presynaptically inhibits glutamate release in rat hippocampus.

Ciproxifan is an H3 receptor antagonist and inverse agonist with antipsychotic effects in several preclinical models; its effect on glutamate release has been investigated in the rat hippocampus. In a synaptosomal preparation, ciproxifan reduced 4-aminopyridine (4-AP)-evoked Ca2+-dependent glutamate release and cytosolic Ca2+ concentration elevation but did not affect the membrane potential. The inhibitory effect of ciproxifan on 4-AP-evoked glutamate release was prevented by the Gi/Go-protein inhibitor pertussis toxin and Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was not affected by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Furthermore, the phospholipase A2 (PLA2) inhibitor OBAA, prostaglandin E2 (PGE2), PGE2 subtype 2 (EP2) receptor antagonist PF04418948, and extracellular signal-regulated kinase (ERK) inhibitor FR180204 eliminated the inhibitory effect of ciproxifan on glutamate release. Ciproxifan reduced the 4-AP-evoked phosphorylation of ERK and synapsin I, a presynaptic target of ERK. The ciproxifan-mediated inhibition of glutamate release was prevented in synaptosomes from synapsin I-deficient mice. Moreover, ciproxifan reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that ciproxifan, acting through the blockade of Gi/Go protein-coupled H3 receptors present on hippocampal nerve terminals, reduces voltage-dependent Ca2+ entry by diminishing PLA2/PGE2/EP2 receptor pathway, which subsequently suppresses the ERK/synapsin I cascade to decrease the evoked glutamate release.

Epigenotype, genotype, and phenotype analysis of patients in Taiwan with Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder predisposing to tumorigenesis that results from abnormal expression or function of imprinted genes of chromosome 11p15.5. METHODS: Forty-seven patients in Taiwan with clinical suspicion of BWS were referred for diagnostic testing based on methylation profiling of H19-associated imprinting center (IC) 1 and KCNQ1OT1-associated IC2 using high-resolution melting analysis, multiplex ligation-dependent probe amplification, or high-resolution quantitative methylation profiling. RESULTS: Twenty-eight patients received a clinical diagnosis of BWS (the presence of 3 major features or 2 major features and at least 1 minor feature), 18 had suspected BWS (the presence of at least 1 major feature), and 1 had isolated Wilms' tumor. Nineteen patients were identified with IC2 hypomethylation (including 1 with isolated Wilms' tumor), 1 with IC1 hypermethylation, 2 with paternal uniparental disomy, and 1 with CDKN1C mutation. Several clinical features were found to be statistically different (P<0.05) between the 2 groups-clinical diagnosis of BWS (n=28) or suspected BWS (n=18)-including macroglossia, pre- or postnatal gigantism, abdominal wall defect, ear creases, facial nevus flammeus, BWS score, and the molecular diagnosis rate. Molecular lesion was detected in 81% of patients with the presence of three major features, compared with 33% and 28% of those with two or one major feature, respectively. The mean BWS score was 5.6 for 19 subjects with "IC2 hypomethylation", compared with 3.8 for 2 subjects with pUPD. The BWS score of one subject with CDKN1C mutation and one with IC1 hypermethylation was 6 and 7, respectively. CONCLUSIONS: The BWS score was positively correlated with the molecular diagnosis rate (P<0.01). The BWS database of epigenotype, genotype, and phenotype is expected to promote better genetic counseling and medical care of these patients.

Cardiac structure and function and effects of enzyme replacement therapy in patients with mucopolysaccharidoses I, II, IVA and VI.

BACKGROUND: While enzyme replacement therapy (ERT) has been shown to improve endurance and joint mobility for patients with mucopolysaccharidoses (MPS) I, II, IVA and VI, the impact of ERT on cardiac abnormalities remains uncertain. METHODS: Medical records and echocardiograms of 28 Taiwanese MPS patients (9 with MPS I, 7 with MPS II, 7 with MPS IVA, and 5 with MPS VI) treated with ERT for 1-10.8years were retrospectively reviewed. RESULTS: At start of ERT, z scores>2 were identified in 46% and 75% for left ventricular mass index (LVMI) and interventricular septum thickness in diastole (IVSd) in these patients, respectively. Twenty-four patients (86%) had valvular heart disease. After ERT, the mean IVSd z score of all patients decreased significantly from 3.87 to 2.57 (p=0.016). For 11 patients starting ERT before 12years of age, z scores for both LVMI and IVSd decreased significantly (p<0.01) after ERT. However, the condition of valve regurgitation or stenosis did not show improvement despite ERT. CONCLUSIONS: ERT was shown to be an effective therapy for reducing cardiac hypertrophy, with best results seen when ERT was started at an early age. ERT, however, had little impact on valvular heart disease.

Hesperidin inhibits glutamate release and exerts neuroprotection against excitotoxicity induced by kainic acid in the hippocampus of rats.

The citrus flavonoid hesperidin exerts neuroprotective effects and could cross the blood-brain barrier. Given the involvement of glutamate neurotoxicity in the pathogenesis of neurodegenerative disorders, this study was conducted to evaluate the potential role of hesperidin in glutamate release and glutamate neurotoxicity in the hippocampus of rats. In rat hippocampal nerve terminals (synaptosomes), hesperidin inhibited the release of glutamate and elevation of cytosolic free Ca(2+) concentration evoked by 4-aminopyridine (4-AP), but did not alter 4-AP-mediated depolarization. The inhibitory effect of hesperidin on evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking the activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels or protein kinase C. In hippocampal slice preparations, whole-cell patch clamp experiments showed that hesperidin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, indicating the involvement of a presynaptic mechanism. In addition, intraperitoneal (i.p.) injection of kainic acid (KA, 15 mg/kg) elevated the extracellular glutamate levels and caused considerable neuronal loss in the hippocampal CA3 area. These KA-induced alterations were attenuated by pretreatment with hesperidin (10 or 50 mg/kg, i.p.) before administering the KA. These results demonstrate that hesperidin inhibits evoked glutamate release in vitro and attenuates in vivo KA-induced neuronal death in the hippocampus. Our findings indicate that hesperidin may be a promising candidate for preventing or treating glutamate excitotoxicity related brain disorders such as neurodegenerative diseases.

Apigenin, a natural flavonoid, inhibits glutamate release in the rat hippocampus.

The purpose of this study was to examine the effect and mechanism of apigenin, a natural flavonoid, on glutamate release in the rat hippocampus. In rat hippocampal nerve terminals (synaptosomes), apigenin inhibited glutamate release and the elevation of the cytosolic free Ca(2+) concentration evoked by 4-aminopyridine, whereas it had no effect on 4-aminopyridine-mediated depolarization and Na(+) influx. The apigenin-mediated inhibition of evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel activity. Furthermore, we determined that gamma-aminobutyric acid type A (GABAA) receptors are present in the hippocampal nerve terminals because they are colocalized with the presynaptic marker synaptophysin. However, the effect of apigenin on 4-aminopyridine-evoked glutamate release from synaptosomes was unaffected by the GABAA receptor antagonists SR95531 and bicuculline. Furthermore, in slice preparations, whole-cell patch-clamp experiments showed that apigenin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, suggesting a presynaptic mechanism. On the basis of these results, we suggested that apigenin exerts its presynaptic inhibition probably by reducing Ca(2+) entry mediated by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, thereby inhibiting glutamate release from the rat hippocampal nerve terminals.

Myricetin inhibits the release of glutamate in rat cerebrocortical nerve terminals.

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of myricetin, a naturally occurring flavonoid with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. The release of glutamate was evoked by the K(+) channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay the synaptosomal plasma membrane potential, and a Ca(2+) indicator Fura-2 to monitor cytosolic Ca(2+) concentrations ([Ca(2+)]C). Results show that myricetin inhibited 4-AP-evoked glutamate release, and this effect was prevented by chelating extracellular Ca(2+) ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate had no effect on myricetin action. Myricetin did not alter the synaptosomal membrane potential, but decreased 4-AP-induced increases in the cytosolic free Ca(2+) concentration. Furthermore, the myricetin effect on 4-AP-evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release. These results suggest that myricetin inhibits glutamate release from cerebrocortical synaptosomes by attenuating voltage-dependent Ca(2+) entry. This implies that the inhibition of glutamate release is an important pharmacological activity of myricetin that may play a critical role in the apparent clinical efficacy of this compound.

Generalized bullous fixed drug eruption is distinct from Stevens-Johnson syndrome/toxic epidermal necrolysis by immunohistopathological features.

BACKGROUND: Generalized bullous fixed drug eruption (GBFDE), a particular form of fixed drug eruption (FDE), is characterized by widespread blisters and erosions and can be confused with Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). OBJECTIVE: We sought to analyze specific features of GBFDE and differentiate it from SJS/TEN. METHODS: We retrospectively studied patients with GBFDE and SJS/TEN during a period of 10 years. GBFDE was defined as typical FDE lesions with blisters involving at least 10% body surface area on at least 3 of 6 different anatomic sites. Clinical presentations; histopathological features; immunohistochemical patterns of cluster-of-differentiation (CD)3, CD4, CD8, CD56, Fas, Fas ligand, granzyme B, perforin, granulysin, and forkhead box P3 (Foxp3); and serum granulysin levels were compared. RESULTS: Twenty-three cases of GBFDE were collected. Patients with GBFDE had shorter latent periods, less mucosal involvement, more eosinophil infiltration, and dermal melanophages. Lesional infiltrates in GBFDE had more dermal CD4(+) cells including Foxp3(+) regulatory T cells, fewer intraepidermal CD56(+) cells, and fewer intraepidermal granulysin(+) cells. The serum level of granulysin in GBFDE was also significantly lower than in SJS/TEN. LIMITATIONS: The number of cases in this study is small. CONCLUSION: GBFDE is a distinct disease distinguishable from SJS/TEN by particular features such as granulysin, CD56, and Foxp3 expressions.