[Human umbilical mesenchymal stem cells-derived exosomes modulate the proliferation, apoptosis and migration of human retinal pigment epithelial cells in hypoxia].

Objective: To study the effects of human umbilical mesenchymal stem cells (HUMSCs) exosomes on the proliferation and apoptotic as well as migration of human retinal pigment epithelial cells (HRPE) in hypoxia, and explore its mechanism. Method: Direct adherent culture was adopted to cultivate umbilical cord mesenchymal stem cells and amplified to the fourth generation. Markers on the cell surface were identified by flow cytometry. Culture medium was collect without serum from the 4th generation umbilical cord mesenchymal stem cells. Exosomes were separated and extracted, then the ultrastructure was observed under electron microscope and examined expression of CD63 and CD9 protein by Western blot method with isolated and extracted exosomes. HRPE was cultivated in vitro culture, proliferation was detected at the time point of 0, 1, 2, 3, 4, 5 d with MTT assay under hypoxic condition. Meanwhile, the cell migration was quantified by Wound-Healing Assay under hypoxic condition at 0, 24, 48 and 72 h respectively combined with apoptosis test. The HRPE cells in the growth period were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml). In all groups, apoptosis was observed by Annexin V/PI dual-dye flow cytometry after 48 h's incubation. Proliferation was observed by MTT assay and the migration was observed with Wound-Healing Assay. Results: Flow cytometry detection of the surface marker of HUMSCs in the 4th generation showed strong positive expression of CD105, CD73, CD90. It was suggested that HUMSCs with isolated culture had MSC specific phenotype with duction of lipids and osteoblasts in vitro. The separated exosomes were observed with spherical membranous structures in different sizes by scanning electron microscopy, and Western blot detected positive expression of CD63 and CD9. In vitro culture of HRPE detected by MTT assay for cell proliferation at the time of hypoxic 0, 1, 2, 3, 4, 5 d, the results showed that, comparing with time point 0 d, other groups had statistically significant OD values. In the first 2 days, the proliferation ability of RPE cells gradually increased as the time of hypoxia prolonged(1.862±0.135, 2.278±0.244). After 3 d, the proliferation ability of RPE cells gradually decreased(1.419±0.124, 1.599±0.156). Wound-Healing Assay results showed that the migration distance gradually increased as[(29.883±4.504), (36.200±1.928) µm] the time of hypoxia increased from 0 to 72 h. The cells were fully covering at the point of 72 h [(1.223±0.194), (0.430±0.299) µm]. Apoptosis test results showed that the number of apoptotic cells was different(3.628%±1.348%, 20.123%±1.183%) with the extension of hypoxia Oxygen before 2 d from 0 to 72 h. At the time of d3, there were more apoptotic cells(42.290%±3.217%). There is a significant difference from pre-2d.RPE cells were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml).After 48 h hypoxia incubation, MTT assay results showed that, compared with the control group (1.870±0.499), the number of cell proliferation was significantly increased (t=-3.116, P<0.05), while compared with the hypoxia group(2.616±0.307), the proliferation number of exosomes was significantly reduced [(2.041±0.115), (1.931±0.205), (1.929±0.025); t=-4.920, -4.540, -5.286, P<0.01], and there was no significant difference between groups with different doses of the exosomes (F=1.181,P>0.05). Annexin V/PI dual-dye flow cytometry was used to observe the apoptosis results. Compared with the control group 1.180%±0.689%, the number of apoptosis in hypoxia group was significantly increased (19.273%±1.194%, t=-32.141, P<0.01), while compared with the hypoxia group, the number of apoptosis in the exosomes was significantly decreased (12.318%±1.087%, 11.878%±1.348%, 11.090%±1.716%; t=-10.547, -10.057, 9.589, P<0.01). There was no significant difference between the groups with different doses of exosomes (F=1.173, P>0.05). Wound-Healing Assay results showed that, compared with the control group(68.047±2.851) µm, the migration distance of the hypoxia group was significantly increased [(13.470±2.255)µm, t=36.778, P<0.01] while compared with the hypoxia group, the migration distance of the exosomes was reduced (33.110±1.774, 24.650±1.175, 26.440±1.674; t=11.766, 10.770, 11.311, P<0.01), and there was no significant difference between the groups of the exosomes (F=1.179, P>0.05). Conclusion: Human umbilical cord mesenchymal stem cells can effectively inhibit the apoptosis and migration of HRPE cells in hypoxia. It provides a theoretical basis for the research and treatment of RPE related diseases. (Chin J Ophthalmol, 2019, 55: 933-941).

[Application of gene capture technology on mutation screening of RB1 gene in retinoblastoma patients].

Objectives: To analyze RB1 gene mutation in retinoblastoma (RB) patients using gene capture technology. Methods: Experimental research. The clinical data of 17 RB patients were collected at Department of Ophthalmology, Peking University People's Hospital from June 2010 to Jun 2014. Peripheral blood samples of seventeen RB patients and their parents were collected and genomic DNA were extracted. DNA library from RB patients was mixed with designed gene capture probe of RB1 exons and its flanking sequences. The data were analyzed using bioinformatics software. To avoid the false positive, the abnormal sites were verified using the Sanger sequencing method. Results: Totally, there were 17 RB patients, including 12 males and 5 females, from 0.5 to 23 years old, average ages were (3.2±5.2) years old. Both eyes were involved in 6 patients. The other 11 cases were only one eye was attacked. Four RB patients were found to have germline mutations, among whom 2 had bilateral tumors and 2 had unilateral tumors. 2 novel missense mutations were identified, including 15(th) exon c.1408A>T (p. Ile470Phe) and c.1960G>C (p. Val654Leu) at 19(th) exon. No RB1 mutation was identified in any of their parents. We also identified 2 mutations reported previously. One is c.1030C>T termination mutation at 10(th) exon in a bilateral RB patients and his father, who was diagnosed with unilateral RB. The other is c.371-372delTA frame shift mutation at 3(rd) exon. No mutation was found in their parents. Conclusions: Two novel germline RB1 mutations were found using gene capture technology, which enriched RB1 mutations library.(Chin J Ophthalmol, 2017, 53: 455-459).

Toll-like receptor 3 polymorphism is not associated with neovascular age-related macular degeneration and polypoidal choroidal vasculopathy in the Chinese.

Toll-like receptor 3 (TLR3) variants in mainland northern Chinese patients with polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (nAMD) were investigated. The complete genes of TLR3, including all exons and the promoter region, were assessed using direct sequencing technology of 284 unrelated mainland northern Chinese individuals: 96 nAMD patients, 92 PCV patients, and 96 controls. Six single nucleotide polymorphisms were identified: rs5743303, rs5743305, rs5743312, rs3775291, rs3775290, and rs6830345. The distribution of TLR3 genotypes for nAMD and PCV was not significantly different compared with normal controls. This study indicates that the TLR3 gene polymorphism is not associated with nAMD and PCV in northern Chinese patients.

Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells.

Excessive oxidative stress in pancreatic ß cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells

Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

Meta-analysis of the relationship between the LOC387715/ARMS2 polymorphism and polypoidal choroidal vasculopathy.

We investigated the association between the LOC387715/ARMS2 polymorphism (rs10490924 G>T) and susceptibility to polypoidal choroidal vasculopathy (PCV) through a meta-analysis of 1446 cases and 3255 controls from eight case-control studies. The genetic effect of the LOC387715/ARMS2 rs10490924 G>T polymorphism on PCV was assessed by calculating pooled odds ratios (ORs) with 95% confidence intervals (95%CIs). We found that elevated PCV risk was significantly associated with the GG genotype (GG vs TT, OR = 4.23, 95%CI = 3.53-5.06), and heterozygous genotype TG appeared to have a minor effect on PCV risk (TG vs TT, OR = 1.47, 95%CI = 1.26-1.71). Patients with the T allele were 2.09 times more likely to have PCV than those with the G allele (95%CI = 1.906-2.288). A further subgroup analysis by ages also showed that the genetic effect of the LOC387715/ARMS2 rs10490924 G>T polymorphism on PCV is stronger among patients with mean age <73 years. Our meta-analysis strengthened the evidence that the LOC387715/ARMS2 rs10490924 G>T polymorphism plays an important role in PCV susceptibility.

Effects of siRNA-targeting BMP-2 on the abilities of migration and invasion of human liver cancer SMMC7721 cells and its mechanism.

To observe the effects of small interfering RNA (siRNA)-targeting bone morphogenetic protein (BMP)-2 on the abilities of migration and invasion of human liver cancer SMMC7721 cells and its mechanism. Three siRNAs-targeting BMP-2 gene were synthesized. There were six groups including group I (non-transfected cells), group II (only liposome-transfected cells), group III (non-specific siRNA-transfected cells) and groups IV-VI (siRNA-A, siRNA-B and siRNA-C-targeting BMP-2 transfected cells, respectively). SMMC7721 cells were instantaneously transfected using lipofectamine method. The levels of mRNA and protein of BMP-2 in cells were determined with reverse transcription-PCR and western blotting. The abilities of migration and invasion of transfected cells were assessed using scratch test and in vitro invasion assay, respectively. The protein levels of p-ERK, p-JNK and p-p38 and the protein levels of MMP-2 and MMP-9 were evaluated with western blot 48 h after siRNA-B-targeting BMP-2 was transfected into liver cancer SMMC7721 cells. Expression of mRNA and protein of BMP-2 in groups IV-VI were significantly inhibited, especially in group V. Cell scratch width was significantly greater in group V than in group I and III (P<0.01). In vitro invasion assay suggested that the number of invasion of cells was significantly lower in group V than in group I and III (P<0.05). Western blot indicated that the level of p-ERK was significantly decreased (P<0.05), the levels of p-JHK and p-p38 were not significantly changed and the levels of MMP-2 and MMP-9 were significantly downregulated (P<0.05). siRNA-targeting BMP-2 can markedly inhibited the expression of BMP-2 in liver cancer SMMC7721 cells, and decrease the abilities of migration and invasion of liver cancer cells, especially siRNA-B. The inhibitory effects of siRNA-B-targeting BMP-2 on the abilities of migration and invasion of human liver cancer SMMC7721 cells may be caused by the downregulation of MMP-2 and MMP-9 through MAPK/ERK pathway, whereas is not related to MAPK/JNK and MAPK/p38 pathway.

Expression of nicotinic acetylcholine receptors and subunit messenger RNAs in the enteric nervous system of the neonatal rat.

In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the CNS and peripheral nervous system. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand (125)I-epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (alpha2, alpha3, alpha4, alpha5, alpha6) and structural beta (beta2, beta3, beta4) subunits. The results showed strong nicotine sensitive binding of (125)I-epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of alpha3, alpha5, alpha7, beta2 and beta4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of alpha3, alpha5 and beta4 was found in some ganglia. There was no signal for alpha4, alpha6 and beta3 in the ENS but positive hybridization signal for alpha2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of alpha3(alpha5)beta4, and a few alpha3beta2 nAChRs. In addition, homomeric alpha7 nAChRs could be present.

Acute nicotine activates c-fos and activity-regulated cytoskeletal associated protein mRNA expression in limbic brain areas involved in the central stress-response in rat pups during a period of hypo-responsiveness to stress.

In adult rats, acute nicotine, the major psychoactive ingredient in tobacco smoke, stimulates the hypothalamic-pituitary-adrenal axis (HPA), resulting in activation of brain areas involved in stress and anxiety-linked behavior. However, in rat pups the first two postnatal weeks are characterized by hypo-responsiveness to stress, also called the 'stress non-responsive period' (SNRP). Therefore, we wanted to address the question if acute nicotine stimulates areas involved in the stress response during SNRP. To determine neuronal activation, the expression of the immediate-early genes c-fos and activity-regulated cytoskeletal associated protein (Arc) was studied in the central nucleus of the amygdala (CeA), bed nucleus stria terminalis (BST) and paraventricular hypothalamic nucleus (PVN), which are areas involved in the neuroendocrine and central stress response. Rat pups received nicotine tartrate (2 mg/kg) or saline by i.p. injection at postnatal days (P) 5, 7 and 10 and their brains were removed after 30 min. We used semi-quantitative radioactive in situ hybridization with gene specific antisense cRNA probes in coronal sections. In control pups, c-fos expression was low in most brain regions, but robust Arc hybridization was found in several areas including cingulate cortex, hippocampus and caudate. Acute nicotine resulted in significant induction of c-fos expression in the PVN and CeA at P5, P7 and P10, and in the BST at P7 and P10. Acute nicotine significantly induced expression of Arc in CeA at P5, P7 and P10, and in the BST at P10. In conclusion, acute nicotine age dependently activated different brain areas of the HPA axis during the SNRP. After P7, the response was more pronounced and included the BST, suggesting differential maturation of the HPA axis in response to nicotine.

Effects of chronic neonatal nicotine exposure on nicotinic acetylcholine receptor binding, cell death and morphology in hippocampus and cerebellum.

Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric alpha7 nAChRs, neuroprotection to heteromeric alpha4beta2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for alpha4, alpha7 and beta2 mRNA was determined by in situ hybridization; nAChR binding sites by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl Violet-stained sections. In control cerebellum, strong expression of alpha4, beta2 mRNA and heteromeric nAChRs labeled with [125I]-epibatidine was found in granule cells, and alpha7 mRNA and homomeric nAChRs labeled with [125I]-alpha-bungarotoxin were in the external germinal layer. In control hippocampus, low expression of alpha4 mRNA and heteromeric nAChRs and high expression of alpha7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.

Nicotine regulates mRNA expression of feeding peptides in the arcuate nucleus in neonatal rat pups.

Maternal smoking results in low birth weight. Using a neonatal gastric intubation model corresponding to the third trimester in humans, nicotine, the major psychoactive ingredient in tobacco, causes growth retardation in rat pups. Here, we wanted to determine the underlying mechanisms of nicotine's anorexic effects. In adults, body weight and energy expenditure are regulated by the adiposity hormone leptin and the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP) and anorexic peptides proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) expressed in the hypothalamic arcuate (Arc) nucleus. Activation of nicotinic acetylcholine receptors (nAChRs) could regulate leptin release and/or peptide expression in the Arc. Neonatal rat pups were treated twice daily with nicotine (0.25, 1.5, and 3 mg/kg) from postnatal day 1 to 8 (P1-8). This resulted in an upregulation of heteromeric nAChR binding sites in the ventromedial nucleus of the hypothalamus and Arc. Nicotine at all three doses significantly reduced body weight gain and increased mRNA expression of NPY, AgRP, and POMC effects, which were blocked by dihydro-beta-erythroidine (DHbetaE), an alpha4beta2* nAChR antagonist, but CART expression was unaffected. In contrast, serum leptin levels were significantly increased only by 3 and 1.5 mg/kg, and the increase was only partially blocked by DHbetaE. These data suggest that in neonates chronic nicotine regulates body weight gain independent from serum leptin levels by a central mechanism involving alpha4beta2* heteromeric nAChRs and stimulated increased expression of the anorexic peptide POMC. Whereas, increased NPY and AgRP expression could be a secondary response to reduction in weight gain.

Chronic neonatal nicotine upregulates heteromeric nicotinic acetylcholine receptor binding without change in subunit mRNA expression.

Smoking during pregnancy chronically exposes the fetus to nicotine resulting in long-term behavioral and cognitive deficits. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated ion channels widely expressed in the nervous system. Chronic nicotine upregulates high-affinity nAChRs in animals and smokers. Here we determined if chronic nicotine treatment during a developmental period corresponding to the human third trimester regulates nAChR expression. Rat pups were intubated orally three times per day with or without nicotine (6 mg/kg/day) from postnatal day 1 to 8. Subunit mRNA expression was assessed by in situ hybridization. Expression of heteromeric and homomeric nAChR receptor was evaluated by autoradiography using (125)I-epibatidine and (125)I-alphabungarotoxin, respectively. nAChR expression was analyzed in cortex, hippocampus, thalamus and medial habenula from autoradiograms using computer assisted image analysis. Nicotine induced significant upregulation of heteromeric but not homomeric nAChRs in hippocampus, cortex and thalamus without changes in subunit mRNA expression. No effect of chronic nicotine on receptor expression was detected in the medial habenula, suggesting that nicotine's effect was mainly on alpha4beta2-type heteromeric nAChRs. The nicotine-induced upregulation was reversed after nicotine withdrawal. Receptor blockade by DHbetaE, an antagonist for heteromeric alpha4/beta2 nAChRs, did not prevent upregulation but increased expression to a similar degree as nicotine. Combination of both drugs had a cumulative effect. Thus, although transient, intermittent nicotine exposure as seen in smoking mothers is sufficient to upregulate heteromeric nAChRs during a critical period of brain development and could contribute to the behavioral deficits found in children whose mother smoked.

[Influence of cleftpalate on middle ear conduction and eustachian function].

OBJECTIVE: To explore the influence and its degree of cleftpalate on middle ear conduction and eustachian function. METHOD: 41 cleftpalate patients (82 ears) were performed with otopharyngeal examinations, form of the ostium pharyngeum tubae auditivae observations and acoustic impedance measurements. The results were compared with healthy persons of normal hearing. RESULT: In the cases with cleftpalate, tympanic membrane pathological change incidence was 89.0% (73/82), ostium pharyngeum tubae auditivae of cracked shape was 59.5% (25/42), abnormal tympanogram was 83.1% (64/77), the negative rate of stapedius muscle reflex was 84.4% (65/77). There are significant different in comparison with healthy persons. In synthetic analyses, the rate of secretory otitis media with varied degree in the cleftpalate cases was 74.0% (57/77). CONCLUSION: There was a high incidence of middle ear pathological change and secretory otitis media in the cleftpalate patients. This high incidence was due to susceptivity of the nasopharynx on the infection, weak strength of the dilator tubae eustachii and the deformed shape of the ostium pharyngeum tubae auditivae.

[Calcium and chronic diseases].

Calcium plays a role in physiological functions in our body. Many diseases are associated with physiological dysfunction caused by abnormal calcium homeostasis. In this review, we discussed the relationships between calcium and some common chronic diseases including cardiovascular diseases. Here we put emphasis on hypertension and atherosclerosis, tumor, osteoporosis and osseous hyperostosis. The most appropriate daily calcium intake and adverse effects of high-calcium diets are also mentioned in this article.

A pilot study on the changes of event-related potentials in school-aged children with iron deficiency anaemia.

Event-related potentials (ERP) were assessed in 70 school-aged children with a diagnosis of asymptomatic iron deficiency anaemia (IDA), based on low haemoglobin and either low serum ferritin or high free erythrocyte protoporphyrin levels. The IDA subjects were randomized into treatment and placebo groups of 35 cases each, and compared with a normal control group of 30 age- and gender-matched healthy subjects without iron deficiency. Further haematological and ERP assessment was carried out after 3 months, during which time the active group received iron supplementation with 10 mg ferrous sulphate, together with vitamin C, malic acid and folic acid. Pre-treatment, both IDA groups had prolonged P300 latencies in comparison with the non-IDA controls (p<0.01). The proportion of cases with distorted wave appearance was more than twice as high in the IDA groups as in the non-IDA controls, although intergroup differences did not reach statistical significance. After treatment, the active treatment IDA group showed a significant increase in haemoglobin levels and shortening in P300 latencies. After treatment, neither value was statistically different from non-IDA controls. There was a decrease in the number of cases with abnormal waveforms in the active treatment group, compared with an increase in the number within the placebo group (P = 0.002). Testing of ERP shows promise as a non-invasive, sensitive and objective marker for assessing cognitive impairment in children with IDA.

Variables affecting electronic root canal measurement.

This study was conducted in two parts. In the first part, 20 single-rooted teeth that had been scheduled for extraction were investigated. The electronic root canal lengths were measured in vivo with a Dental Sono-Explorer type Y-III, and the actual canal lengths were measured after extraction of the teeth. The rate of agreement of the two measurements was 77.5% within a range of +/- 0.5 mm, while it was 100% at +/- 2.0 mm, which is acceptable clinically. In the second part, there were 19 simulated canals whose lengths and apical foramen sizes were known beforehand. Experiments revealed a negative correlation between the areas of the apical foramina and the difference between the electronic and the actual root canal lengths. This relationship was shown by the linear regression equation: y = 0.6-1.6x. With the exception of the smallest areas of foramina, electronic root canal length measurements were less than the actual lengths.

[Synthesis of muscopyridine and its analogs].

Muscopyridine (1) is one of the odoriferous constituents of natural musk. 1 was prepared previously in very low yield in multisteps. We report here one-step preparation of muscopyridine and its analogs by the cross-coupling method. The cyclocoupling is effected by slow dropwise addition of a THF solution of di-Grignard reagent under nitrogen to a well stirred mixture of an equimolar quantity of a dichloropyridine in THF containing a catalytic amount of Ni (dppp)Cl2. The reaction mixture is stirred at 35-40 degrees C for up to 20 h. Analogs 2, 3 and 4 have also been prepared by this method. The structure of these products and intermediates are determined by UV, IR, MS, and 1HNMR spectral data.