[Determination of seven paraben preservatives in aquatic seasoning using solid-phase extraction coupled with high performance liquid chromatography].

Seven parabens are widely used in soy sauce, vinegar, jam, oyster sauce, stuffing, and other foods. The long-term intake of large amounts of parabens and similar substances may be harmful to the human body. Therefore, the addition of paraben preservatives to food should be strictly controlled. The current detection method is applicable to single target compound and several food categories, and the experimental pretreatment method involves extraction with anhydrous ethyl ether, which is a toxic reagent. Moreover, interferences in the analysis of parabens via gas chromatography limit the versatility and accuracy of the detection method. Herein, a novel method based on solid-phase extraction (SPE) coupled with high performance liquid chromatography (HPLC) was developed for the determination of seven paraben preservatives (methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, isopropyl p-hydroxybenzoate, isobutyl p-hydroxybenzoate, and heptyl p-hydroxybenzoate) in oyster sauce, shrimp sauce, and fish sauce. Compared with the conventional method, the proposed work enables the determination of more compounds, thereby expanding its scope of application to different food types. This strategy also optimizes the pretreatment method and device parameters. The samples were extracted with methanol and 20% methanol aqueous solution by ultrasonication, respectively, and then centrifuged. The experimental pretreatment method was enriched, and sample clean-up was conducted using a MAX SPE column. The seven parabens were separated using a Chromcore 120 C18 column (150 mm×4.6 mm, 3.0 µm). Gradient elution was performed with acetonitrile-5 mmol/L ammonium acetate aqueous solution as the mobile phase (initial mobile phase volume ratio, 30∶70). The flow rate was 0.7 mL/min, and the column temperature was 35 ℃. A diode array detector with a detection wavelength of 254 nm was also used. The seven paraben preservatives showed good linearity in the range of 0.5-50.0 mg/L, with correlation coefficients greater than 0.9999. The limits of detection (LODs) and quantification (LOQs) for the seven paraben preservatives were 0.2-0.4 mg/kg and 0.5-1.3 mg/kg, respectively. A spiked recovery test was conducted using oyster sauce, shrimp sauce, and fish sauce at three spiked levels of 2, 40, and 200 mg/kg. Good recoveries for the seven paraben preservatives were obtained and the recoveries of the analytes in oyster sauce, shrimp sauce, and fish sauce were 91.0%-102%, 95.5%-106%, and 95.0%-105%, respectively, with relative standard deviations of ≤6.97%. Compared with the liquid-liquid extraction method, the proposed method demonstrated better purification effects. The recoveries of the seven paraben preservatives extracted using this method were also much higher than those obtained from liquid-liquid extraction. We determined the contents of these preservatives in 135 food products using the method established in this study and detected methyl p-hydroxybenzoate and ethyl p-hydroxybenzoate in soy sauce, vinegar, and pickles. Thus, the established method can be used for the effective determination of seven parabens in aquatic seasoning such as oyster sauce, shrimp sauce, and fish sauce.

Glycyrrhizin regulates the HMGB1/P38MAPK signalling pathway in status epilepticus.

In recent decades, studies have reported that inflammation serves key roles in epilepsy and that high mobility group box protein­1 (HMGB1) may be involved in status epilepticus. However, it has not been reported whether HMGB1 participates in the pathogenesis of status epilepticus through the regulation of the p38 mitogen­activated protein kinase (p38MAPK) signalling pathway. In the present study, Sprague­Dawley rats were randomly divided into four groups as follows: Control, status epilepticus (SE), dimethyl sulfoxide treatment (DMSO + SE), and glycyrrhizin treatment (GL + SE) groups. Behavioural changes were then evaluated using the Racine score. In the hippocampus, the protein expression levels of HMGB1 were assessed using western blotting, the neuronal damage was evaluated using haematoxylin and eosin staining and transmission electron microscopy, and the activation of microglia was assessed using immunochemistry and immunofluorescence. The results demonstrated that, in the hippocampal region, HMGB1 existed in neurons and astrocytes and the protein expression levels of HMGB1, p38MAPK and phosphorylated­p38MAPK were significantly inhibited after treatment with GL. Furthermore, GL could alleviate neuronal injury in the CA1 region of the hippocampus and prevented HMGB1 translocation from the nucleus into the cytoplasm in these areas. These findings expand the understanding of how HMGB1 may participate in SE and lay a foundation for evaluation of HMGB1 as a drug target.

Mechanism of Robo1 in the pentylenetetrazol-kindled epilepsy mouse model.

The neural network hypothesis is one of the important pathogenesis of drug-resistant epilepsy. Axons guide molecules through synaptic remodeling and brain tissue remodeling, which may result in the formation of abnormal neural networks. Therefore, axon guidance plays a crucial role in disease progression. However, although Robo1 is one of the important components of axon guidance, the role of Robo1 in epilepsy remains unclear. In this study, we aimed to explore the mechanism of Robo1 in epilepsy. Male adult C57BL/6 mice were intraperitoneally injected with pentylenetetrazol to establish an epilepsy model. Lentivirus (LV) was given via intracranial injection 2 weeks before pentylenetetrazol injection. Different expressions of Robo1 between the control group, LV-mediated Robo1 short hairpin RNA group, empty vector control LV group, and normal saline group were analyzed using Western blot, immunofluorescence staining, Golgi staining, and video monitoring. Robo1 was increased in the hippocampus in the pentylenetetrazol-induced epilepsy mouse model; lentiviral Robo1 knockdown prolonged the latency of seizure and reduced the seizure grade in mice and resulted in a decrease in dendritic spine density, while the number of mature dendritic spines was maintained. We speculate that Robo1 has been implicated in the development and progression of epilepsy through its effects on dendritic spine morphology and density. Epileptic mice with Robo1 knockdown virus intervention had lower seizure grade and longer latency. Follow-up findings suggest that Robo1 may modulate seizures by affecting dendritic spine density and morphology. Downregulation of Robo1 may negatively regulate epileptogenesis by decreasing the density of dendritic spines and maintaining a greater number of mature dendritic spines.

Novel mutation of EPM2A causes progressive myoclonic epilepsy: a case report.

We report a case of progressive myoclonic epilepsy caused by a novel mutation in EPM2A. The female patient experienced abnormal jerky movements of the involving all four limbs and several generalized seizures, degeneration of cognition, and unsteadiness. Genetic analysis identified two rare, deleterious mutations in exon4: chr6: 145,948,751(c.G797G > A) and chr6: 145,948,761(c.T787C > T). The mutations at these two loci were from the genomes of their mother and father, respectively, which were compound heterozygous variations. This report updates the mutation sites of gene EPM2A and extends genotype-phenotype correlations in Lafora disease.

Characterization of Rhizosphere and Endophytic Microbial Communities Associated with Stipa purpurea and Their Correlation with Soil Environmental Factors.

This study was to explore the diversity of rhizosphere and endophytic microbial communities and the correlation with soil environmental factors of Stipa purpurea on the Qinghai-Tibetan Plateau. The bacterial phylum of Proteobacteria, Firmicutes and Bacteroidota, and the fungal phylum of Ascomycota, Basidiomycota and Zygomycota were dominant in microbial communities of S. purpurea in all three sampling sites. Multiple comparison analysis showed that there were significant differences in the composition of microbial communities in the roots, leaves and rhizosphere soil. Whether it is fungi or bacteria, the OTU abundance of rhizosphere soils was higher than that of leaves and roots at the same location, while the difference among locations was not obvious. Moreover, RDA analysis showed that Zygomycota, Cercozoa, Glomeromycota, Chytridiomycota and Rozellomycota possessed strongly positive associations with altitude, dehydrogenase, alkaline phosphatase, neutral phosphatase, available kalium and available phosphate, while Ascomycota was strongly negatively associated. Changes in ammonium nitrate, alkaline phosphatase, polyphenol oxidase, total phosphorus, and altitude had a significant impact on the bacterial communities in different habitats and altitudes. Taken together, we provide evidence that S. purpurea has abundant microbial communities in the alpine grassland of the Qinghai-Tibetan Plateau, whose composition and diversity are affected by various soil environmental factors.

Effects of Allelochemicals, Soil Enzyme Activities, and Environmental Factors on Rhizosphere Soil Microbial Community of Stellera chamaejasme L. along a Growth-Coverage Gradient.

Allelochemicals released from the root of Stellera chamaejasme L. into rhizosphere soil are an important factor for its invasion of natural grasslands. The aim of this study is to explore the interactions among allelochemicals, soil physicochemical properties, soil enzyme activities, and the rhizosphere soil microbial communities of S. chamaejasme along a growth-coverage gradient. High-throughput sequencing was used to determine the microbial composition of the rhizosphere soil sample, and high-performance liquid chromatography was used to detect allelopathic substances. The main fungal phyla in the rhizosphere soil with a growth coverage of 0% was Basidiomycetes, and the other sample plots were Ascomycetes. Proteobacteria and Acidobacteria were the dominant bacterial phyla in all sites. RDA analysis showed that neochamaejasmin B, chamaechromone, and dihydrodaphnetin B were positively correlated with Ascomycota and Glomeromycota and negatively correlated with Basidiomycota. Neochamaejasmin B and chamaechromone were positively correlated with Proteobacteria and Actinobacteria and negatively correlated with Acidobacteria and Planctomycetes. Allelochemicals, soil physicochemical properties, and enzyme activity affected the composition and diversity of the rhizosphere soil microbial community to some extent. When the growth coverage of S. chamaejasme reached the primary stage, it had the greatest impact on soil physicochemical properties and enzyme activities.

Molecular properties and structural characterization of an alkaline extractable arabinoxylan from hull-less barley bran.

An alkaline extractable arabinoxylan (HBAX-25) was fractionated from crude arabinoxylan (HBAX) obtained optimally in hull-less barley (Hordeum vulgare L. var. nudum Hook. f.) bran. Molecular properties and structural characterization of HBAX-25 were investigated thoroughly based on chemical composition of 8.31% (w/w) moisture and 87.57% (w/w) sugar with specifically few proteins (1.08%, w/w) and high arabinoxylans (82.46%, w/w). Data from monosaccharide composition indicated that HBAX-25 mainly consisted of arabinose (30.13 mol%) and xylose (51.55 mol%) with A/X ratio of 0.58, representative for arabinoxylans, which coincided with FT-IR results and was corroborated by methylation and NMR analyses, i.e., a relatively low-branched arabinoxylan composed of un-substituted (1,4-linked ß-D-Xylp, 71.19%), mono-substituted (1,3,4-linked ß-D-Xylp, 14.78%) and di-substituted (1,2,3,4-linked ß-D-Xylp, 10.76%) xylose units as backbone via ß-(1→4) linkages, with six possible branches or individuals included. Hence, a structural basis of HBAX-25 was established, which could have potential in food and other value-added applications capable of interpreting their physicochemical, functional and technological characteristics.

Interactions between GHRH and GABAARs in the brains of patients with epilepsy and in animal models of epilepsy.

Growth hormone releasing hormone (GHRH) has recently been shown to increase the level of γ-aminobutyric acid (GABA) and activate GABA receptors (GABARs) in the cerebral cortex. GABA is an inhibitory neurotransmitter that can inhibit seizures. Does GHRH enhance the inhibitory effect of GABA to prevent epilepsy by increasing the GABA level and activating GABARs? In this study, patients with epilepsy and C57/BL6 mice with epilepsy induced by kainic acid (KA) or pentylenetetrazol (PTZ) served as the research subjects. Western blots were used to observe the differences in GHRH expression between the normal group and the epilepsy group, immunofluorescence was performed to explore the localization of GHRH in the brain, and coimmunoprecipitation was used to observe the interaction between GHRH and GABARs. GHRH expression was significantly increased in both patients with temporal lobe epilepsy (TLE) and in two mouse models induced by KA or PTZ compared with that in the normal groups (P < 0.05 or P < 0.01). GHRH was expressed in neurons in both humans and mice. Additionally, GHRH co-localized with presynaptic and postsynaptic sites of inhibitory neurons. Coimmunoprecipitation confirmed that GHRH interacted with GABAAα1 and GABAAß2 + 3. GHRH may play an important role in inhibiting seizures by activating GABAARs.

Nitrobenzylthioinosine mimics adenosine to attenuate the epileptiform discharge of hippocampal neurons from epileptic rats.

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn't change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50µM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.

[The observation and analysis the function and morphology of the eustachian tube in secretory otitis media and chronic rhinosinusitis in children].

OBJECTIVE: To observe and analyze the function and morphology of pharyngeal ostium of the eustachian tubes in secretory otitis media and chronic rhinosinusitis in children under direct vision,in order to provide an objective basis for clinical treatments. METHOD: Fifty cases of secretory otitis media,50 cases of chronic rhinosinusitis and a control group of 50 cases with hoarseness were examined under video laryngoscope to observe the pharyngeal ostium morphological changes of the eustachian tubes, and their functional statuses were tested by using acoustic impedance instrument. All the data were analyzed by statistical methods. RESULT: (1) In the secretory otitis group, the abnomal rate of the pharyngeal ostium of the eustachian tubes was 94% while the chronic rhinosinusitis group was 80%,and between them there was no significant differences (P > 0.05). But both of them had significant differences with the control group (P < 0.05). (2) In the secretory otitis group, the rate of the eustachian tube dysfunction was 70% while the chronic rhinosinusitis group was 26%, and between them there was significant differences (P < 0.05), and both of them have significant differences when compared with the control group (P < 0.05). CONCLUSION: There are some abnormal points exist in the function and the morphology of the eustachian tube in secretory otitis media and chronic rhinosinusitis in children. Eustachian tube dysfunction played a dominant role in the pathogenesis of secretory otitis media in children rather than the morphological change did compared to the chronic rhinosinusitis in children.

Effects of valproate sodium on extracellular signal-regulated kinase 1/2 phosphorylation following hippocampal neuronal epileptiform discharge in rats.

The aim of the present study was to investigate the effects of valproate sodium (VPAS) on the phosphorylation extracellular signal-regulated kinase 1/2 (ERK1/2) following hippocampal neuronal epileptiform discharge in rat neurons. The study used neurons from female and male neonate Sprague-Dawley (SD) rats (at least 24 h old), which were rapidly decapitated. Following the successful development of the epileptiform discharge cell model, the neurons were divided into two groups, the VPAS group and the control group. In the concentration-response experiment, the neurons were incubated with three different concentrations of VPAS (50, 75 and 100 mg/l) 30 min prior to the epileptiform discharge. The expression of phosphorylated ERK1/2 (p-ERK1/2) was examined using an immunofluorescence technique. In the time-response experiment, the neurons were incubated with VPAS (50 mg/l) and monitored at different time-points (30 min prior to the epileptiform discharge and 0 min, 30 min, 2 h and 6 h subsequent to epileptiform discharge), and western blotting was employed to measure the changes in p-ERK1/2 protein expression. No significant differences in the expression of p-ERK1/2 among the neurons treated with different concentrations of VPAS were identified in the concentration-response experiment. However, in the time-response experiment, the expression of p-ERK1/2 30 min prior to the epileptiform discharge was significantly lower compared with that at the other time-points. Furthermore, 50 mg/l VPAS was capable of decreasing the action potential frequency of the neuronal epileptiform discharge. ERK1/2 was excessively and persistently activated following the epileptiform discharge of the neurons. In addition, a low concentration of VPAS was effective at inhibiting the phosphorylation of ERK1/2 at an earlier period of neuronal epileptiform discharge.

Subarachnoid hemorrhage associated with epidemic hemorrhagic fever: a rare case report.

Nervous system injuries associated with epidemic hemorrhagic fever (EHF) are not rarely seen. However, cerebrovascular disease arising from EHF is rarely reported in the literature. A 50-year-old male patient suffered from subarachnoid hemorrhage (SAH). No abnormal condition was found in intracranial vascular digital subtraction angiography (DSA). But, this patient presented with positive hantavirus-IgM and IgG, with typical clinical process, which lead to the diagnosis of EHF followed by SAH. To our knowledge, SAH associated with EHF has not been previously reported. A meticulous assessment of EHF patients with a serious condition had one or more central nervous system (CNS) abnormalities, such as sudden headache, vomiting, confusion, meningismus, and convulsions, which is necessary for diagnosing and giving timely treatment to improve the prognosis.