Raster plots machine learning to predict the seizure liability of drugs and to identify drugs.

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However, there are still issues surrounding the analysis methods used to predict seizure and toxicity liability as well as drug mechanisms of action. In the present study, we developed an artificial intelligence (AI) capable of predicting the seizure liability of drugs and identifying drugs using deep learning based on raster plots of neural network activity. The seizure liability prediction AI had a prediction accuracy of 98.4% for the drugs used to train it, classifying them correctly based on their responses as either seizure-causing compounds or seizure-free compounds. The AI also made concentration-dependent judgments of the seizure liability of drugs that it was not trained on. In addition, the drug identification AI implemented using the leave-one-sample-out scheme could distinguish among 13 seizure-causing compounds as well as seizure-free compound responses, with a mean accuracy of 99.9 ± 0.1% for all drugs. These AI prediction models are able to identify seizure liability concentration-dependence, rank the level of seizure liability based on the seizure liability probability, and identify the mechanism of the action of compounds. This holds promise for the future of in vitro MEA assessment as a powerful, high-accuracy new seizure liability prediction method.

Electrophysiological responses to seizurogenic compounds dependent on E/I balance in human iPSC-derived cortical neural networks.

Construction of in vitro functional assay systems using human-induced pluripotent stem cells (iPSCs) as indicators for evaluating seizure liability of compounds has been anticipated. Imbalance of excitation/inhibition (E/I) inputs triggers seizure; however, the appropriate ratio of E/I neurons for evaluating seizure liability of compounds in a human iPSC-derived neural network is unknown. Here, five neural networks with varying E/I ratios (88/12, 84/16, 74/26, 58/42, and 48/52) were constructed by altering the ratios of glutamatergic (E) and GABA (I) neurons. The responsiveness of each network against six seizurogenic compounds and two GABA receptor agonists was then examined by using six representative parameters. The 52% GABA neuron network, which had the highest ratio of GABA neurons, showed the most marked response to seizurogenic compounds, however, it suggested the possibility of producing false positives. Moreover, analytical parameters were found to vary with E/I ratio and to differ for seizurogenic compounds with different mechanism of action (MoA) even at the same E/I ratio. Clustering analysis using six parameters showed the balance of 84/16, which is the closest to the biological balance, was the most suitable for detection of concentration-dependent change and classification of the MoA of seizurogenic compounds. These results suggest the importance of using a human-iPSC-derived neural network similar to the E/I balance of the living body in order to improve the prediction accuracy in the in vitro seizure liability assessment.

Analysis of signal components < 500 Hz in brain organoids coupled to microelectrode arrays: A reliable test-bed for preclinical seizure liability assessment of drugs and screening of antiepileptic drugs.

Brain organoids with three-dimensional structure and tissue-like function are highly demanded for brain disease research and drug evaluation. However, to our knowledge, methods for measuring and analyzing brain organoid function have not been developed yet. This study focused on the frequency components of an obtained waveform below 500 Hz using planner microelectrode array (MEA) and evaluated the response to the convulsants pentylenetetrazol (PTZ) and strychnine as well as the antiepileptic drugs (AEDs) perampanel and phenytoin. Sudden and persistent seizure-like firing was observed with PTZ administration, displaying a concentration-dependent periodic activity with the frequency component enhanced even in one oscillation characteristic. On the other hand, in the administration of AEDs, the frequency of oscillation decreased in a concentration-dependent manner and the intensity of the frequency component in one oscillation also decreased. Interestingly, at low doses of phenytoin, a group of synchronized bursts was formed, which was different from the response to the perampanel. Frequency components contained information on cerebral organoid function, and MEA was proven useful in predicting the seizure liability of drugs and evaluating the effect of AEDs with a different mechanism of action. In addition, frequency component analysis of brain organoids using MEA is an important analysis method to perform in vitro to in vivo extrapolation in the future, which will help explore the function of the organoid itself, study human brain developments, and treat various brain diseases.

HIGH BACKGROUND AREA FOR RADIATION EDUCATION.

This paper describes our trial experience of the use of high radiation area for radiation education. We used environmental samples collected from the high radiation area in Fukushima prefecture and India, for the practice of radiation measurement and health risk assessment in Nagasaki University Medical School. We also carried out the field monitoring seminar for students in the existing exposure areas in Tottori prefecture and the Yamakiya observatory in Fukushima. Although the evaluation of educational effectiveness is still underway, both types of education appeared attractive for the students mostly due to the exposure from natural environment in our real life which was not achieved by using an artificial radiation source in a classroom.

Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system.

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However, differences in responsiveness depending on convulsant type and antiepileptic drugs, and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ, but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses, frequency analysis of below 250 Hz, excluding the spike component, was performed. The in vivo convulsive firing enhancement of the high γ wave and ß wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods, including frequency analysis, appear effective for predicting convulsion toxicity, side effects, and their mechanism of action as well as the comparison of convulsions induced in vivo.

Detection of synchronized burst firing in cultured human induced pluripotent stem cell-derived neurons using a 4-step method.

Human induced pluripotent stem cell-derived neurons are promising for use in toxicity evaluations in nonclinical studies. The multi-electrode array (MEA) assay is used in such evaluation systems because it can measure the electrophysiological function of a neural network noninvasively and with high throughput. Synchronized burst firing (SBF) is the main analytic parameter of pharmacological effects in MEA data, but an accurate method for detecting SBFs has not been established. In this study, we present a 4-step method that accurately detects a target SBF confirmed by the researcher's interpretation of a raster plot. This method calculates one set parameter per step, in the following order: the inter-spike interval (ISI), the number of spikes in an SBF, the inter-SBF interval, and the number of spikes in an SBF again. We found that the 4-step method is advantageous over the conventional method because it determines the preferable duration of an SBF, accurately distinguishes continuous SBFs, detects weak SBFs, and avoids false detection of SBFs. We found also that pharmacological evaluations involving SBF analysis may differ depending on whether the 4-step or conventional threshold method is used. This 4-step method may contribute to improving the accuracy of drug toxicity and efficacy evaluations using human induced pluripotent stem cell-derived neurons.

Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture.

The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20-30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

Induction of long-term potentiation and depression phenomena in human induced pluripotent stem cell-derived cortical neurons.

Plasticity such as long-term potentiation (LTP) and long-term potentiation depression (LTD) in neuronal networks has been analyzed using in vitro and in vivo techniques in simple animals to understand learning, memory, and development in brain function. Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for understanding the plasticity mechanism in human neuronal networks, thereby elucidating disease mechanisms and drug discoveries. In this study, we attempted the induction of LTP and LTD phenomena in a cultured hiPSC-derived cerebral cortical neuronal network using multi-electrode array (MEA) systems. High-frequency stimulation (HFS) produced a potentiated and depressed transmission in a neuronal circuit for 1 h in the evoked responses by test stimulus. The cross-correlation of responses revealed that spike patterns with specific timing were generated during LTP induction and disappeared during LTD induction and that the hiPSC-derived cortical neuronal network has the potential to repeatedly express the spike pattern with a precise timing change within 0.5 ms. We also detected the phenomenon for late-phase LTP (L-LTP) like plasticity and the effects for synchronized burst firing (SBF) in spontaneous firings by HFS. In conclusion, we detected the LTP and LTD phenomena in a hiPSC-derived neuronal network as the change of spike pattern. The studies of plasticity using hiPSC-derived neurons and a MEA system may be beneficial for clarifying the functions of human neuronal circuits and for applying to drug screening.

The role performance of public health nurses as clinical instructors in Japan.

AIM: To investigate the factors associated with the role performance of public health nurses as clinical instructors in Japan. BACKGROUND: Newly graduated public health nurses in Japan have competencies that are below the minimum requirements of the Ministry of Health, Labour and Welfare because of their limited clinical experience in undergraduate clinical education. Public health nurses play crucial roles in the clinical practicum and their role performance as clinical instructors is a key to successful learning outcomes. METHODS: This study targeted public health nurses in governmental public health centres and those who had gained experience as an undergraduate clinical instructor for nursing students. A self-administered questionnaire was distributed to a national sample of 1467 public health nurses. Data were collected from July 2011 to September 2011. RESULTS: In total, 722 of 1467 questionnaires were completed (nurse age 22-64 years). Of the participants, almost half (49%) strongly disagreed (3%) or disagreed (46%) that they had confidence in their role as a clinical instructor, and preparation programmes for clinical instructors had been attended by just 262 (36.3%). Years of experience as public health nurses, previous attendance of preparation programmes, viewing their role positively, professional identity and professional competency were significantly associated with performance. Logistic regression analysis revealed that nurses with higher role performance scores had higher self-confidence, greater interests in their role and higher professional identity. CONCLUSIONS: The self-confidence and interests of public health nurses in their role as clinical instructors as well as their professional identity were found to be significant predictors of their role performance as clinical instructors. IMPLICATIONS FOR NURSING PRACTICE AND EDUCATION: The factors identified in our investigation can be used to predict effective clinical instructors and to develop preparation programmes to enhance their confidence and interests and potentially increase their role satisfaction.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for species identification of Acinetobacter strains isolated from blood cultures.

The clinical relevance of Acinetobacter species, other than A. baumannii, as human pathogens has not been sufficiently assessed owing to the insufficiency of simple phenotypic clinical diagnostic laboratory tests. Infections caused by these organisms have different impacts on clinical outcome and require different treatment and management approaches. It is therefore important to correctly identify Acinetobacter species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been introduced to identify a wide range of microorganisms in clinical laboratories, but only a few studies have examined its utility for identifying Acinetobacter species, particularly those of the non-Acinetobacter baumannii complex. We therefore evaluated MALDI-TOF MS for identification of Acinetobacter species by comparing it with sequence analysis of rpoB using 123 isolates of Acinetobacter species from blood. Of the isolates examined, we identified 106/123 (86.2%) to species, and 16/123 (13.0%) could only be identified as acinetobacters. The identity of one isolate could not be established. Of the 106 species identified, 89/106 (84.0%) were confirmed by rpoB sequence analysis, and 17/106 (16.0%) were discordant. These data indicate correct identification of 89/123 (72.4%) isolates. Surprisingly, all blood culture isolates were identified as 13 species of Acinetobacter, and the incidence of Acinetobacter pittii was unexpectedly high (42/123; 34.1%) and exceeded that of A. baumannii (22/123; 17.9%). Although the present identification rate using MALDI-TOF MS is not acceptable for species-level identification of Acinetobacter, further expansion of the database should remedy this situation.

Microbial contamination of a disinfectant-soaked unwoven cleaning cloth.

In December 2009, a 76-year-old male patient developed pneumonia due to Burkholderia cepacia whilst in an intensive care unit at a Japanese university hospital. During the subsequent environmental investigation to find the source, B. cepacia with an identical DNA type was found in his denture storage solution. Open packets of unwoven rayon cloths soaked in 0.2% alkyldiaminoethylglycine hydrochloride, used for environmental cleaning, were shown to be contaminated with B. cepacia, Alcaligenes xylosoxidans, Pseudomonas fluorescens and Pseudomonas aeruginosa. B. cepacia of a different DNA type was found in five of 42 samples from sealed packets of cloths.

Roles of neuronal nitric oxide synthase, oxidative stress, and propofol in N-methyl-D-aspartate-induced dilatation of cerebral arterioles.

BACKGROUND: It remains unclear whether N-methyl-D-aspartate (NMDA) receptors contribute to cerebral parenchymal vasodilatation, and any effects of clinically used anaesthetics on the dilatation. The present study was designed to examine whether NMDA induces neuronal nitric oxide synthase (NOS)-mediated dilatation, in the cerebral parenchymal arterioles, and whether propofol and superoxide modulate the dilatation in relation to the NMDA receptor activation. METHODS: The cerebral parenchymal arterioles within rat brain slices were monitored by a computer-assisted microscopy, and the vasodilatation in response to NMDA (10(-7) to 10(-5) M) was evaluated. Immunofluorescence analysis to neuronal and endothelial NOS and measurement of levels of superoxide and nitric oxide within the arteriole were simultaneously performed. RESULTS: Propofol, an NMDA receptor antagonist MK801, and a neuronal NOS antagonist S-methyl-l-thiocitrulline (SMTC) reduced NMDA-induced dilation, whereas a superoxide inhibitor, Tiron, and NADPH oxidase inhibitor, gp91ds-tat, augmented NMDA-induced dilatation. Immunofluorescence analysis revealed distribution of neuronal NOS in both endothelial and smooth muscle cells in addition to neuronal cells. NMDA-induced superoxide and nitric oxide within the parenchymal arterioles. The increased superoxide within the arteriole was similarly inhibited by MK801, SMTC, gp91ds-tat, propofol, and a neuronal NOS antagonist vinyl-l-NIO, whereas the level of nitric oxide was reduced by MK801, SMTC, propofol, and vinyl-l-NIO, and it was augmented by gp91ds-tat. CONCLUSIONS: NMDA dilates cerebral parenchymal arterioles possibly via neuronal NOS activation, whereas it produces superoxide via NADPH oxidase. In these arterioles, propofol reduces both the dilatation and superoxide production in response to NMDA.

PP091. Prematurity and hypertensive disorders in pregnancy. A major public health problem.

INTRODUCTION: Around the world each year 10% to 15% of direct maternal deaths are associated with hypertensive disorders in pregnancy. Not only it can be devastating and life threatening for the mothers but also for the babies. Risks for the baby include poor growth and prematurity [1]. In low and middle income countries many public hospitals have limited access to neonatal intensive care, and so the mortality and morbidity is likely to be considerably higher than in settings where such facilities are available. Thus, the approach to the problem preeclampsia should include issues related to prematurity and its impact, since many of these babies develop definitive complications, constituting a major public health problem with social and economic repercussions. OBJECTIVES: To evaluate the prevalence of preterm births in a public hospital health of the city of São Paulo, checking its main causal conditions, focusing in particular on the importance of hypertensive disorders in its determinism. METHODS: This was a retrospective study of a consecutive series of preterm infants, defined as gestational age less than 37 weeks born at Maternity School Vila Nova Cachoeirinha, situated at the north of Sao Paulo City, in the period from 01/04 to 31/12/2011. The study population was classified according to three main groups of causal factors: spontaneous labor, premature rupture of membranes and elective preterm delivery. In this group was evaluated the specific participation of hypertensive disorders. RESULTS: The number of live births during the study period was 5302 babies. Among these 433 had gestational age less than 37 weeks, corresponding to a prematurity rate of 8.16%. Of all infants, 385 cases were included in the analysis. Regarding the causes of the onset of preterm labor found that 140 cases (36.4%) presented with spontaneous labor, 128 cases (33.2%) had premature rupture of membranes and 117 (30.4%) cases were born as a result of elective preterm delivery. In the latter group 88 cases (75.2%) had complications related to hypertensive disorders. Regarding the general population of premature infants, hypertension accounted for 22.8% of cases. CONCLUSION: We conclude that among the various obstetric problems, hypertensive disorders represent an important impact on preterm birth in our setting, considering that our institution is a reference to this type of care across the city of São Paulo. This knowledge is a fundamental tool to support the adoption of interventions that can detect groups at risk for hypertension in pregnancy, the promotion of follow-up to intercept severe cases and provide an efficient network of maternity care that may have neonatal intensive care units. This set of measures is essential to minimize the impact of this serious problem.

Role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in gastric ulcer healing in mice.

We examined the role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in the healing of gastric ulcers in mice. Male M-CSF-deficient (op/op) and M-CSF-expressing heterozygote (+/?) mice were used. Gastric ulcers were induced by thermal cauterization under ether anesthesia, and healing was observed for 14 days after ulceration. The numbers of macrophages and microvessels in the gastric mucosa were determined immunohistochemically with anti-CD68 and anti-CD31 antibodies, respectively. Expression of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF) mRNA was determined via real-time reverse transcription-polymerase chain reaction (RT-PCR), and the mucosal content of prostaglandin (PG) E(2) was determined via enzyme immunoassay on day 10 after ulceration. The healing of gastric ulcers was significantly delayed in op/op mice compared with +/? mice. Further, significantly fewer macrophages were observed in the normal gastric mucosa of op/op mice than in +/? mice. Ulcer induction caused a marked accumulation of macrophages around the ulcer base in +/? mice, but this response was attenuated in op/op mice. The mucosal PGE(2) content as well as the expression of COX-2, VEGF, and TNF-α mRNA were all upregulated in the ulcerated area of +/? mice but significantly suppressed in op/op mice. The degree of vascularization in the ulcerated area was significantly lower in op/op mice than in +/? mice. Taken together, these results suggest that M-CSF-dependent macrophages play an important role in the healing of gastric ulcers, and that this action may be associated with angiogenesis promoted by upregulation of COX-2/PGE(2) production.

Investigation of the presence of ghrelin in the central nervous system of the rat and mouse.

Ghrelin and ghrelin receptor agonist have effects on central neurons in many locations, including the hypothalamus, caudal brain stem, and spinal cord. However, descriptions of the distributions of ghrelin-like immunoreactivity in the CNS in published work are inconsistent. We have used three well-characterized anti-ghrelin antibodies, an antibody to the unacylated form of ghrelin, and a ghrelin peptide assay in rats, mice, ghrelin knockout mice, and ghrelin receptor reporter mice to re-evaluate ghrelin presence in the rodent CNS. The stomach served as a positive control. All antibodies were effective in revealing gastric endocrine cells. However, no specific staining could be found in the brain or spinal cord. Concentrations of antibody 10 to 30 times those effective in the stomach bound to nerve cells in rat and mouse brain, but this binding was not reduced by absorbing concentrations of ghrelin peptide, or by use of ghrelin gene knockout mice. Concentrations of ghrelin-like peptide, detected by enzyme-linked immunosorbent assay in extracts of hypothalamus, were 1% of gastric concentrations. Ghrelin receptor-expressing neurons had no adjacent ghrelin immunoreactive terminals. It is concluded that there are insignificant amounts of authentic ghrelin in neurons in the mouse or rat CNS and that ghrelin receptor-expressing neurons do not receive synaptic inputs from ghrelin-immunoreactive nerve terminals in these species.

Molecular analysis of new isolates of Tomato leaf curl Philippines virus and an associated betasatellite occurring in the Philippines.

Three new begomovirus isolates and one betasatellite were obtained from a tomato plant exhibiting leaf curl symptom in Laguna, the Philippines. Typical begomovirus DNA components representing the three isolates (PH01, PH02 and PH03) were cloned, and their full-length sequences were determined to be 2754 to 2746 nucleotides. The genome organizations of these isolates were similar to those of other Old World monopartite begomoviruses. The sequence data indicated that PH01 and PH02 were variants of strain B of the species Tomato leaf curl Philippines virus, while PH03 was a variant of strain A of the species Tomato leaf curl Philippines virus. These isolates were designated ToLCPV-B[PH:Lag1:06], ToLCPV-B[PH:Lag2:06], and ToLCPV-A[PH:Lag3:06], respectively. Phylogenetic analysis revealed that the present isolates form a separate monophyletic cluster with indigenous begomoviruses reported earlier in the Philippines. A betasatellite isolated from same sample belongs to the betasatellite species Tomato leaf curl Philippines betasatellite and designated Tomato leaf curl Philippines betasatellite-[Philippines:Laguna1:2006], ToLCPHB-[PH:Lag1:06]. When co-inoculated with this betasatellite, tomato leaf curl Philippines virus induced severe symptoms in N. benthamiana and Solanum lycopersicum plants. Using a PVX-mediated transient assay, we found that the C4 and C2 proteins of tomato leaf curl Philippines virus and the ßC1 protein of ToLCPHB-[PH:Lag1:06] function as a suppressor of RNA silencing.

Cerebral oxygenation in the beach chair position before and during general anesthesia.

AIM: Ischemic brain damage related to the beach chair position is a matter of concern. The current study was designed to evaluate whether the beach chair position before and during general anesthesia differentially induces changes in cerebral oxygenation as determined by near-infrared spectroscopy (NIRS) in surgical patients. METHODS: We evaluated brain tissue oxygen index (TOI) values using the NIRS monitor NIRO-200TM in the beach chair position the day before and during general anesthesia. Thirty patients with normal preoperative TOI values undergoing shoulder surgery were enrolled. The initial TOI measurement in the supine position after 10 min rest or 10 min after tracheal intubation was followed by measurements after 5 min each in the 30-degree and subsequently 60-degree head-up tilt positions. During general anesthesia, patients were mechanically ventilated to obtain normocapnia under inhalation of 1.5% sevoflurane in 50% oxygen. Mean blood pressure (MAP) was measured non-invasively in the arm at heart level and was maintained above 60 mmHg with phenylephrine. RESULTS: Preoperative TOI values and preoperative MAP were within the normal range in the study population. MAP decreased upon anesthesia but did not further change when the patient was placed in the 30- and 60-degree head-up tilt positions. Heart rate also decreased upon anesthesia. However, TOI values did not change with induction of general anesthesia or placement of the patients in the beach chair position. CONCLUSION: Under general anesthesia, the beach chair position does not alter cerebral oxygenation in patients showing normal preoperative cerebral TOI values.