Cell chip device for real-time monitoring of drug release from drug-laden microparticles.

A cell chip is a microfluidic cell culture device fabricated using microchip manufacturing methods for culturing living cells in a micrometer-sized chamber to model the physiological functions of tissues and organs. It has been extensively investigated in the domain of drug transport and toxicity research. Herein, we developed a cell chip for real-time monitoring of drug release from drug carriers. The proposed system integrates three core functions: cell culture, real-time analysis, and drug delivery tests. This device was designed to be loaded with microparticles for drug release and to enable real-time drug measurement. The efficacy of the developed system was evaluated by measuring the concentration of drugs released from the microparticles prepared with poly(lactic-co-glycolic acid) (PLGA). Doxorubicin, an anticancer drug, was used as a model drug and A549 cells, a type of lung cancer cell, were simultaneously cultured to compare the drug release concentrations in the presence of cells. Furthermore, variations in cell viability with respect to the presence of drug-loaded microparticles were observed and analyzed. Notably, as the proposed system requires an extremely small number of microparticles, it affords simple implementation in a single device, thereby eliminating the need for complex accessories and instruments for analysis. Thus, the analysis process becomes more convenient and cost-efficient. Thus, the proposed method offers an easy analysis of the release behavior of various cells and drugs. The simplicity and low cost of this innovative system without sacrificing analytical precision demonstrate its potential for applications across various fields.

Photocoagulation Up to Ora Serrata in Diabetic Vitrectomy to Prevent Recurrent Vitreous Hemorrhage.

PURPOSE: To evaluate the role of performing photocoagulation up to ora serrata during vitrectomy in preventing recurrent vitreous hemorrhage (VH) in patients undergoing pars plana vitrectomy (PPV) for proliferative diabetic retinopathy (PDR). METHODS: This retrospective, nonrandomized study included 60 eyes from 60 patients who had undergone PPV for VH due to PDR. These patients were divided into two groups: group 1, those who underwent photocoagulation up to ora serrata using the scleral indentation technique during surgery; and group 2, those who did not undergo scleral indentation when photocoagulation and underwent photocoagulation up to vortex veins. Their hospital records were analyzed to investigate the recurrence rate of VH, the time until recurrence of VH after surgery, logarithm of the minimal angle of resolution (logMAR) best-corrected visual acuity (BCVA) measured before surgery and at 1, 2, and 3 years after surgery, and the occurrence of complications such as neovascular glaucoma (NVG) during follow-up. RESULTS: Group 1 exhibited lower recurrence rate of VH (2 of 30 [6.7%] vs. 10 of 30 [33.3%], p = 0.01) and lower occurrence of postoperative NVG (2 of 30 [6.7%] vs. 8 of 30 [26.7%], p = 0.038) compared with group 2. There were no statistically significant differences in logMAR BCVA measured at 1, 2, and 3 years between the two groups (at 1 year: 0.54 ± 0.43 vs. 0.54 ± 0.44, p = 0.954; at 2 years: 0.48 ± 0.47 vs. 0.55 ± 0.64, p = 0.235; at 3 years: 0.51 ± 0.50 vs. 0.61 ± 0.77, p = 0.200). Logistic regression analysis showed that among several factors that could affect recurrence rate of VH, only range of photocoagulation performed was a statistically significant factor (odds ratio, 0.119; 95% confidence interval, 0.022-0.659; p = 0.015). CONCLUSIONS: Photocoagulation treatment over a wider range with scleral indentation could be a beneficial adjunct procedure for preventing postoperative recurrent VH following diabetic vitrectomy.

Whole body vibration accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

This study aimed to investigate the effect of whole body vibration (WBV) on the sensory and motor nerve components with sciatic nerve injury model rats. Surgery was performed on 21 female Wister rats (6-8 weeks) under intraperitoneal anesthesia. The nerve-crush injuries for the left sciatic nerve were inflicted using a Sugita aneurysm clip. The sciatic nerve model rats were randomly divided into two groups (n=9; control group, n=12; WBV group). The rats in the WBV group walked in the cage with a vibratory stimulus (frequency 50 Hz, 20 min/day, 5 times/wk), while those in the control group walked in the cage without any vibratory stimulus. We used heat stimulation-induced sensory threshold and lumbar magnetic stimulation-induced motor-evoked potentials (MEPs) to measure the sensory and motor nerve components, respectively. Further, morphological measurements, bilateral hind-limb dimension, bilateral gastrocnemius dimension, and weight were evaluated. Consequently, there were no significant differences in the sensory threshold at the injury side between the control and WBV groups. However, at 4 and 6 weeks postoperatively, MEPs latencies in the WBV group were significantly shorter than those in the control group. Furthermore, both sides of the hind-limb dimension at 6 weeks postoperatively, the left side of the gastrocnemius dimension, and both sides of the gastrocnemius weight significantly increased. In conclusion, WBV especially accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

Serous cystic neoplasm: Do we have to wait till it causes trouble? Season 2.

A 50-year-old male presented gradually growing pancreatic body mass. An abdominal computed tomography showed a 9.9-cm mass, larger than the 8.9-cm mass one year ago. As the patient did not have complaints for any symptomatic problems, the gastroenterologist decided to check it with regular follow-up. However, as the tumor grew faster than expected, the patient was recommended for surgical resection. Laparoscopic pylorus preserving pancreaticoduodenectomy was done. Since the tumor abutted to the superior mesenteric vein and the portal vein, wedge resection of vessel was inevitable. Pathology was serous cystadenoma. The patient was discharged without postoperative complications. Herein, we report this case with asymptomatic large serous cystic neoplasm treated by laparoscopic approach. The appropriateness of current guidelines for surgery in serous cystic neoplasm is also discussed.

Early-stage COVID-19 pandemic observations on pulmonary embolism using nationwide multi-institutional data harvesting.

We introduce a multi-institutional data harvesting (MIDH) method for longitudinal observation of medical imaging utilization and reporting. By tracking both large-scale utilization and clinical imaging results data, the MIDH approach is targeted at measuring surrogates for important disease-related observational quantities over time. To quantitatively investigate its clinical applicability, we performed a retrospective multi-institutional study encompassing 13 healthcare systems throughout the United States before and after the 2020 COVID-19 pandemic. Using repurposed software infrastructure of a commercial AI-based image analysis service, we harvested data on medical imaging service requests and radiology reports for 40,037 computed tomography pulmonary angiograms (CTPA) to evaluate for pulmonary embolism (PE). Specifically, we compared two 70-day observational periods, namely (i) a pre-pandemic control period from 11/25/2019 through 2/2/2020, and (ii) a period during the early COVID-19 pandemic from 3/8/2020 through 5/16/2020. Natural language processing (NLP) on final radiology reports served as the ground truth for identifying positive PE cases, where we found an NLP accuracy of 98% for classifying radiology reports as positive or negative for PE based on a manual review of 2,400 radiology reports. Fewer CTPA exams were performed during the early COVID-19 pandemic than during the pre-pandemic period (9806 vs. 12,106). However, the PE positivity rate was significantly higher (11.6 vs. 9.9%, p < 10-4) with an excess of 92 PE cases during the early COVID-19 outbreak, i.e., ~1.3 daily PE cases more than statistically expected. Our results suggest that MIDH can contribute value as an exploratory tool, aiming at a better understanding of pandemic-related effects on healthcare.

Free gait in a shallow pool accelerates recovery after exercise in model mice with fibromyalgia.

This study aimed to determine the effect of pool gait exercise using fibromyalgia-induced model mice. The sensory threshold, locomotive behavior, electrocardiogram, and onset time after the gait test in shallow water using male C57BL/6J mice (weight, 30-35 g; n=21) were investigated. To induce fibromyalgia in model mice, reserpine was injected intraperitoneally into wild-type mice once a day for 3 days. Subsequently, the fibromyalgia-induced model mice were randomly classified into two groups as follows: the control group (n=11) and the pool gait group (n=10). The mice in the pool gait group walked in the same cage containing shallow warm water 5 times per week. Both groups underwent sensory thresholds and video recordings to determine locomotive behaviors weekly. Further, both heart rate and video recordings for observation of a recovery after the gait test in shallow water were undertaken (control group; n=5, pool gait group; n=5). The pool gait did not affect sensory thresholds and locomotive behavior; however, in the pool gait group, both the recovery after the test, such as onset time and gait distance, were considerably better than those of the control group. Furthermore, changes in heart rate and heart rate irregularity after the test were more apparent in the control group than in the pool gait group. The free gait in a shallow pool accelerated recovery after exercise, unlike the sensory threshold.

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model.

BACKGROUND: Fibromyalgia patients experience cardiovascular complications in addition to musculoskeletal pain. This study aimed to investigate the cardiac effects of a prolonged shallow water gait in a fibromyalgia-induced young mouse model. METHODS: To produce a fibromyalgia mouse model, wild-type mice were administered an intraperitoneal injection of reserpine once a day for three days, and two primary experiments were performed. First, three types of gait tests were performed before and after the reserpine injections as follows: (i) 5 minutes of free gait outside the water, (ii) 1 minute of free gait in shallow warm water, and (iii) 5 minutes of free gait in shallow warm water. Second, electrocardiogram recordings were taken before and after the three gait tests. The average heart rate and heart rate irregularity scores were analyzed. RESULTS: Exercise-induced cardiac arrhythmia was observed at 1-minute gait in shallow water during the acute stage of induced FM in young mice. Further, both cardiac arrhythmia and a decrease in HR have occurred at 5-minute gait in shallow water at the same mice. However, this phenomenon was not observed in the wild-type mice under any test conditions. CONCLUSION: Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, we should be aware of the risk of prolonged and excessive exercise-induced cardiac arrhythmia. For gait exercises in shallow water as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted.

Prevalence and risk factors of childhood blepharoptosis in Koreans: the Korea National Health and Nutrition Examination Survey.

PURPOSE: Childhood blepharoptosis may cause cosmetic and functional problems in children, but there is a paucity of studies about its epidemiology. This study aimed to investigate the prevalence of childhood blepharoptosis and associated risk factors in a representative Korean population. METHODS: This cross-sectional nation-wide study analysed the data set acquired from the Korea National Health and Nutrition Examination Survey 2008-2012. A total of 8218 children aged 3-18 years were included. The prevalence of childhood blepharoptosis, defined as a margin reflex distance (MRD) of < 2 mm in either eye, was estimated, and the risk factors were identified using multivariate logistic regression analysis. RESULTS: The mean age of participants was 11.3 ± 0.1 years, and 52.8 ± 0.6% were boys. The overall prevalence of childhood blepharoptosis in Korea was 8.0% (95% CI, 6.9-9.1%). Boys exhibited a higher prevalence of blepharoptosis than girls at most of ages. Levator function increased with age in the normal general population. The proportion of subjects exhibiting MRD1 ≥ 4.0 mm also increased significantly with age (p < 0.001). Male gender, higher body mass index, and urban residency were significantly associated with childhood blepharoptosis. CONCLUSIONS: The prevalence of childhood blepharoptosis is higher in urban obese boys. The increase of levator function with age should be considered in evaluations of childhood ptosis.

Xenon modulates synaptic transmission to rat hippocampal CA3 neurons at both pre- and postsynaptic sites.

KEY POINTS: Xenon (Xe) non-competitively inhibited whole-cell excitatory glutamatergic current (IGlu ) and whole-cell currents gated by ionotropic glutamate receptors (IAMPA , IKA , INMDA ), but had no effect on inhibitory GABAergic whole-cell current (IGABA ). Xe decreased only the frequency of glutamatergic spontaneous and miniature excitatory postsynaptic currents and GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude or decay times of these synaptic responses. Xe decreased the amplitude of both the action potential-evoked excitatory and the action potential-evoked inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) via a presynaptic inhibition in transmitter release. We conclude that the main site of action of Xe is presynaptic in both excitatory and inhibitory synapses, and that the Xe inhibition is much greater for eEPSCs than for eIPSCs. ABSTRACT: To clarify how xenon (Xe) modulates excitatory and inhibitory whole-cell and synaptic responses, we conducted an electrophysiological experiment using the 'synapse bouton preparation' dissociated mechanically from the rat hippocampal CA3 region. This technique can evaluate pure single- or multi-synapse responses and enabled us to accurately quantify how Xe influences pre- and postsynaptic aspects of synaptic transmission. Xe inhibited whole-cell glutamatergic current (IGlu ) and whole-cell currents gated by the three subtypes of glutamate receptor (IAMPA , IKA and INMDA ). Inhibition of these ionotropic currents occurred in a concentration-dependent, non-competitive and voltage-independent manner. Xe markedly depressed the slow steady current component of IAMPA almost without altering the fast phasic IAMPA component non-desensitized by cyclothiazide. It decreased current frequency without affecting the amplitude and current kinetics of glutamatergic spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents. It decreased the amplitude, increasing the failure rate (Rf) and paired-pulse rate (PPR) without altering the current kinetics of glutamatergic action potential-evoked excitatory postsynaptic currents. Thus, Xe has a clear presynaptic effect on excitatory synaptic transmission. Xe did not alter the GABA-induced whole-cell current (IGABA ). It decreased the frequency of GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude and current kinetics. It decreased the amplitude and increased the PPR and Rf of the GABAergic action potential-evoked inhibitory postsynaptic currents without altering the current kinetics. Thus, Xe acts exclusively at presynaptic sites at the GABAergic synapse. In conclusion, our data indicate that a presynaptic decrease of excitatory transmission is likely to be the major mechanism by which Xe induces anaesthesia, with little contribution of effects on GABAergic synapses.

Development of Azo-Based Turn-On Chemical Array System for Hydrazine Detection with Fluorescence Pattern Analysis.

A facile turn-on chemical sensor array was developed for hydrazine detection by means of fluorescence pattern recognition. Taking advantage of the unique properties of the azo group, four different fluorogenic probes, Seoul-Fluor (SF)-Azo 01-04, were designed and prepared. SF-Azo 01-04 displayed fluorescence enhancement of up to 800-fold upon reaction with hydrazine, and all probes exhibited excellent selectivity in the presence of various anions and nucleophiles. By employing the probes in a cellulose paper-based array system, the hydrazine concentration was successfully determined by monitoring the change in fluorescent patterns.

Comparison of systemic conditions at diagnosis between central retinal vein occlusion and branch retinal vein occlusion.

OBJECTIVE: To compare systemic conditions at the time of diagnosis between patients with central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). DESIGN: This study included patients diagnosed with CRVO or BRVO between February 2009 and August 2017 at three branch hospitals of Hallym University Medical Center. Demographic and anthropometric variables, systemic comorbidity profiles, and laboratory findings at diagnosis were collected from a clinical data warehouse system, and were compared between the CRVO and BRVO groups. RESULT: Four hundred and seventeen patients with CRVO and 1,511 patients with BRVO were included. The mean age was 61.8 ± 13.9 years, which was comparable between two groups (P = .332). Female proportion was higher in the BRVO group (55.0%) than in the CRVO group (48.0%; P = .013). Diabetes mellitus (P = .017) and chronic kidney disease (P = .004) were more prevalent in the CRVO group. Serum homocysteine level was abnormally high in 23.5% of CRVO patients and in 8.4% of BRVO patients (P < .001). Blood urea nitrogen and serum creatinine levels were abnormally elevated in more subjects with CRVO (P = .002). CONCLUSION: CRVO is associated with higher prevalence of diabetes mellitus and chronic kidney disease, as well as with elevated serum homocysteine level. These results might suggest a difference between the pathophysiologies of CRVO and BRVO.

Direct Visualization of Continuous Meibum Secretion From the Orifices of Meibomian Glands to the Tear Film.

PURPOSE: To present a new method to directly visualize meibum secretion on the tear film from meibomian gland orifices and show that meibum is continuously secreted between blinking. METHODS: Eighteen patients with dry eye syndrome and 17 healthy subjects were included in the study. We used the Lipiscanner to evaluate the tear film lipid layer. The lipid layer was classified into thick, normal, and thin lipid layer. The lipid layer on the lower tear meniscus of the right eye was observed after a drop of saline solution was applied to the eye. We recorded continuous meibum secretion onto the tear meniscus surface. We calculated the rate of continuous meibum secretion by analyzing videos. Noncontact meibography was performed for meibomian glands in the lower eyelid. The quality of meibum from the 5 orifices at the same area was then scored. RESULTS: The mean continuous meibum secretion rate was 2.7 pL/s in the healthy group and 8.0 pL/s in the dry eye group. The rates were 1.3, 6.7, and 9.4 pL/s in the thin, normal, and thick tear film lipid layer group, respectively. They were 3.4, 3.4, 10.7, and 18.1 pL/s in grade 0, 1, 2, and 3 meibomian gland dropout groups, respectively. The rates were 0.00, 4.7, 10.1, 2.0, and 0.7 pL/s in the normal meibum, yellow without increased viscosity, yellow with increased viscosity, toothpaste, and no meibum groups, respectively. CONCLUSIONS: We showed how to visualize meibum being secreted into the tear film from the meibomian gland orifices, and we were able to observe the continuous secretion of meibum between blinks.

Development of Theragnostic Tool Using NIR Fluorescence Probe Targeting Mitochondria in Glioma Cells.

Because mitochondria are essential organelles for regulating energy homeostasis and intrinsic apoptosis, the perturbation of mitochondrial functions has been considered as an anticancer treatment. In this study, a new near-infrared (NIR) fluorescent probe, SiR-Mito11 was developed as a theragnostic agent for brain tumor by targeting mitochondria. SiR-Mito11 exhibited potential anticancer activity against glioma cells but tolerance in normal neuronal cells. We further confirmed that the selective accumulation of SiR-Mito11 in glioma cells disrupted mitochondria membrane potential, followed by apoptotic cell death.

Validation of Alternative Methods for Detecting Meibomian Gland Dropout Without an Infrared Light System: Red Filter for Simple and Effective Meibography.

PURPOSE: To evaluate the validity and reliability of alternative methods for evaluating meibomian gland (MG) dropout without using an infrared light system: red-filtered or isolated red-channel images (RCIs) of the everted eyelid. METHODS: We evaluated MG dropout in the everted upper and lower eyelids of 125 eyes of 64 patients with good-quality infrared meibography images (IMIs) and color digital photographs with and without a red filter. Red-filtered images (RFIs) were converted to black and white and adjusted for contrast/brightness [adjusted red-filtered images (aRFIs)]. RCIs were computationally isolated from color digital photographs obtained without a red filter. After randomization, the total meiboscore (0-6) was evaluated by 2 independent evaluators (interobserver reliability) masked to the image origin, and again after a 30-day interval (intraobserver reliability). RESULTS: The meiboscores evaluated using the RFI, aRFI, and RCI were strongly positively correlated with those evaluated using the IMI (RFI: ρ = 0.788; aRFI: ρ = 0.735; RCI: ρ = 0.630; all P < 0.001, Spearman correlation analysis). Linear-weighted κ-values (κw) showed substantial agreement between the RFI and IMI (κw = 0.676, 95% CI = 0.594-0.759). The RFI had substantial intraobserver reliability (κw = 0.735, 95% CI = 0.685-0.785) and moderate interobserver reliability (κw = 0.467, 95% CI = 0.371-0.563). Computational adjustment of RFIs did not enhance the validity or reliability, and RCIs had limitations in some cases. CONCLUSIONS: MGs were successfully visualized using a red filter on a slit lamp and showed substantial agreement with visualization using the standard infrared method. Although interobserver reliability was only moderate, this alternative technique may be useful for evaluating MG dropout when an infrared meibography device is not available.

The Inhibitory Effects on Cytotoxicity and Nitric Oxide (NO) of the Nano-Encapsulated Extraction of Lipid-Soluble Green Tea Leaves.

The purpose of this study was to investigate the bioactive substances and extraction conditions of lipid-soluble green tee extract using supercritical CO extraction from green tea leaves growing in Boseong-gun, Jeollanam-do and to demonstrate cell safety and effects on inflammatory response of the nano-encapsulated lipid-soluble green tee extract. The polyphenol content of green tea extract using supercritical CO2 extraction was analyzed. The highest polyphenol was extracted in the condition of 400 bar/50 °C. A total of polyphenol content was 2.47 mg/g. The lipid-soluble vitamin content of supercritical extracts of lipid-soluble green tea was analyzed. Beta-carotene, a vitamin A precursor (4.131 mg/100 g) and vitamin E (537.9 mg/100 g) were detected. ECG content was the highest of catechin of lipid-soluble green tea extract. For cell safety and NO inhibition, human skin cell-derived HaCaT cells and RBL-2H3 mast cells did not have cytotoxicity in 1% of the nano-encapsulated lipid-soluble green tea leaf extract. RAW 264.7 cells did not show cytotoxicity in 5%. NO production was significantly decreased in RAW 264.7 cells in a concentration-dependent manner and within the concentration range. Based on the findings, this study suggested the extraction standards and ingredients of polyphenol according to temperature and pressure in extracting green tea leaf ingredients using supercritical CO2 extraction. This study also objectively demonstrated the safety and anti-inflammatory effect of the nano-encapsulated lipid-soluble green tea leaf extract which is used as functional materials in the cosmetic industry.

Both ipsilateral and contralateral localized vibratory stimulations modulated pain-related sensory thresholds on the foot in mice and humans.

PURPOSE: This study was aimed to investigate the effect of localized vibration on sensory thresholds in mice and humans using a novel quantitative method. PARTICIPANTS AND METHODS: The sensory thresholds of 7-week-old male C57BL/6J mice were measured with four sine-wave electrostimulation frequencies (5, 50, 250, and 2,000 Hz) before and after applying 2-minute vibration to the plantar side of the foot in mice. In human participants (16 males and 16 females; mean age, 21.0±0.8 years), the sensory threshold was measured at 50 Hz before and after applying 2-minute and 5-minute vibrations to the dorsal side of the foot. RESULTS: Application of a 2-minute vibration at either the ipsilateral or contralateral side modulated the sensory thresholds elicited by a 5- or 50-Hz right electrostimulation in mice. In human participants, application of a 5-minute vibration at either the ipsilateral or contralateral side modulated the sensory threshold elicited by 50-Hz right electrostimulation, but had no effect on local skin temperature. These results suggest that the right side of pain-related Aδ fibers (50 Hz) or C fibers (5 Hz) was modulated by the localized ipsilateral or contralateral side of vibratory stimuli, respectively, in mice and humans. CONCLUSION: The ability of contralateral vibration to modify the right sensory thresholds suggests possible involvement of the central nervous system in vibratory modulation.

Comparison of efficacy and toxicity between botulinum toxin subtypes A1 and A2 in cynomolgus macaques.

Botulinum toxin type A (subtype A1) is used as therapeutic agent for some neurological disorders causing spasticity. The toxin products have an upper dosage limit, and their adverse events, such as side effects of diffusion following high-dose administration, have become serious issues. Therefore, a preparation with greater therapeutic efficacy at lower dosages and less diffusion in the body is desired. We have attempted to produce neurotoxin derived from subtype A2 (A2NTX), which has a different amino acid sequence from that of neurotoxin derived from subtype A1. In this study, to investigate whether A2NTX is applicable for treatment, we compared the muscle relaxation effects and the toxicity between A1LL and A2NTX in adult cynomolgus macaques. In the isometric muscle contraction test elicited by 30 Hz tetanus stimulation, the contractions observed in the 0.4 U/site A1LL-treated group were similar in value to those in the 0.13 U/site A2NTX-treated group. In the toxicity test, the 12 and 24 U/kg A1LL- and A2NTX-treated groups all exhibited similar signs of toxicity regarding symptoms, rate of weight loss, and decrease in the length of the right lower leg perimeter. Thus, A2NTX demonstrated approximately 3.0-times higher muscle relaxation activity than A1LL, and their toxicity was equivalent. This study suggested that A2NTX products are more suitable for the treatment of neurological disorders.

Calcium channel subtypes on glutamatergic mossy fiber terminals synapsing onto rat hippocampal CA3 neurons.

The current electrophysiological study investigated the functional roles of high- and low-voltage-activated Ca2+ channel subtypes on glutamatergic small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons. Experiments combining both the "synapse bouton" preparation and single-pulse focal stimulation technique were performed using the conventional whole cell patch configuration under voltage-clamp conditions. Nifedipine, at a high concentration, and BAY K 8644 inhibited and facilitated the glutamatergic excitatory postsynaptic currents (eEPSCs) that were evoked by 0.2-Hz stimulation, respectively. However, these drugs had no effects on spontaneous EPSCs (sEPSCs). Following the use of a high stimulation frequency of 3 Hz, however, nifedipine markedly inhibited eEPSCs at the low concentration of 0.3 µM. Moreover, ω-conotoxin GVIA and ω-agatoxin IVA significantly inhibited both sEPSCs and eEPSCs. Furthermore, SNX-482 slightly inhibited eEPSCs. R(-)-efonidipine had no effects on either sEPSCs or eEPSCs. It was concluded that glutamate release from SMFTs depends largely on Ca2+ entry through N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. The contribution of L-type Ca2+ channels to eEPSCs was small at low-firing SMFTs but more significant at high-firing SMFTs. T-type Ca2+ channels did not appear to be involved in neurotransmission at SMFTs. NEW & NOTEWORTHY Action potential-evoked glutamate release from small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons is regulated by high-threshold but not low-threshold Ca2+ channel subtypes. The functional contribution mainly depends on N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. However, in SMFTs stimulated at a high 3-Hz frequency, L-type Ca2+ channels contributed significantly to the currents. The present results are consistent with previous findings from fluorometric studies of large mossy fiber boutons.

4-Chlorophenol biodegradation facilitator composed of recombinant multi-biocatalysts immobilized onto montmorillonite.

A biodegradation facilitator which catalyzes the initial steps of 4-chlorophenol (4-CP) oxidation was prepared by immobilizing multiple enzymes (monooxygenase, CphC-I and dioxygenase, CphA-I) onto a natural inorganic support. The enzymes were obtained via overexpression and purification after cloning the corresponding genes (cphC-I and cphA-I) from Arthrobacter chlorophenolicus A6. Then, the recombinant CphC-I was immobilized onto fulvic acid-activated montmorillonite. The immobilization yield was 60%, and the high enzyme activity (82.6%) was retained after immobilization. Kinetic analysis indicated that the Michaelis-Menten model parameters for the immobilized CphC-I were similar to those for the free enzyme. The enzyme stability was markedly enhanced after immobilization. The immobilized enzyme exhibited a high level of activity even after repetitive use (84.7%) and powdering (65.8%). 4-CP was sequentially oxidized by a multiple enzyme complex, comprising the immobilized CphC-I and CphA-I, via the hydroquinone pathway: oxidative transformation of 4-CP to hydroxyquinol followed by ring fission of hydroxyquinol.