Nanoelectrochemistry reveals how soluble Aß42 oligomers alter vesicular storage and release of glutamate.

Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by ß-amyloid (Aß) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aß42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aß42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aß amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aß42-induced neuronal hyperactivity and neuronal degeneration.

Mechanical Strain Induces and Increases Vesicular Release Monitored by Microfabricated Stretchable Electrodes.

Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single-cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4-ethylenedioxythiophene) conductive ink, which achieved real-time quantitation of strain-induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.

Homeostasis inside Single Activated Phagolysosomes: Quantitative and Selective Measurements of Submillisecond Dynamics of Reactive Oxygen and Nitrogen Species Production with a Nanoelectrochemical Sensor.

Reactive oxygen and nitrogen species (ROS/RNS) are generated by macrophages inside their phagolysosomes. This production is essential for phagocytosis of damaged cells and pathogens, i.e., protecting the organism and maintaining immune homeostasis. The ability to quantitatively and individually monitor the four primary ROS/RNS (ONOO-, H2O2, NO, and NO2-) with submillisecond resolution is clearly warranted to elucidate the still unclear mechanisms of their rapid generation and to track their concentration variations over time inside phagolysosomes, in particular, to document the origin of ROS/RNS homeostasis during phagocytosis. A novel nanowire electrode has been specifically developed for this purpose. It consisted of wrapping a SiC nanowire with a mat of 3 nm platinum nanoparticles whose high electrocatalytic performances allow the characterization and individual measurements of each of the four primary ROS/RNS. This allowed, for the first time, a quantitative, selective, and statistically robust determination of the individual amounts of ROS/RNS present in single dormant phagolysosomes. Additionally, the submillisecond resolution of the nanosensor allowed confirmation and measurement of the rapid ability of phagolysosomes to differentially mobilize their enzyme pools of NADPH oxidases and inducible nitric oxide synthases to finely regulate their homeostasis. This reveals an essential key to immune responses and immunotherapies and rationalizes its biomolecular origin.

[Nucleosides-based identification model for Fritillariae Cirrhosae Bulbus].

A high performance liquid chromatography( HPLC) method was established for the fast,and precise determination of ten nucleosides in Fritillariae Cirrhosae Bulbus and its counterfeits. Then multivariate statistical analyses,such as clustering analysis,principal component analysis( PCA),and Fisher' s linear discriminant analysis( LDA),were conducted to establish a discriminant function model for an integrated analysis. The results indicated that data acquisition time of a single sample was shortened within 16 min by the HPLC method. In the range of 5-1 000 mg·kg~(-1),the mass concentrations of all nucleosides exhibited good linear relationships with the corresponding peak areas( R2> 0. 999). The spiked recoveries were in the range of 93. 83%-108. 9% with RSDs of0. 12%-1. 3%( n = 5). The limit of quantitation( LOQ) was 0. 98-4. 13 mg·kg~(-1). As revealed by the clustering analysis,Fritillariae Cirrhosae Bulbus and the counterfeits could be discriminated into two clusters based on the content of nucleosides. Fisher's LDA could achieve this discrimination,while PCA dimension reduction failed. The accuracy of the discriminant function model established on the screened characteristic indicators reached 97. 5%. The present study proposed a new identification method of Fritillariae Cirrhosae Bulbus with one-dimensional indicators,which is simple,accurate,and reliable. It can provide a scientific basis for further optimizing the identification techniques for Fritillariae Cirrhosae Bulbus and inspiration for quality control strategy development of Chinese medicinal materials.

Quantitative Nano-amperometric Measurement of Intravesicular Glutamate Content and its Sub-Quantal Release by Living Neurons.

Quantitative measurements of intravesicular glutamate (Glu) and of transient exocytotic release contents directly from individual living neurons are highly desired for understanding the mechanisms (full or sub-quantal release?) of synaptic transmission and plasticity. However, this could not be achieved so far due to the lack of adequate experimental strategies relying on selective and sensitive Glu nanosensors. Herein, we introduce a novel electrochemical Glu nanobiosensor based on a single SiC nanowire that can selectively measure in real-time Glu fluxes released via exocytosis by large Glu vesicles (ca. 125 nm diameter) present in single hippocampal axonal varicosities as well as their intravesicular content before exocytosis. These measurements revealed a sub-quantal release mode in living hippocampal neurons, viz., only ca. one third to one half of intravesicular Glu molecules are released by individual vesicles during exocytotic events. Importantly, this fraction remained practically the same when hippocampal neurons were pretreated with L-Glu-precursor L-glutamine, while it significantly increased after zinc treatment, although in both cases the intravesicular contents were drastically affected.

Aß1-42 Oligomers Induced a Short-Term Increase of Glutamate Release Prior to Its Depletion As Measured by Amperometry on Single Varicosities.

Glutamate (Glu) is a critical neurotransmitter for neuronal communication in the nervous system. In vivo studies have shown that the concentration of Glu is reduced within the brains of those afflicted with Alzheimer's disease (AD), which is also associated with the accumulation of pathogenic amyloid-beta (Aß). However, the effects of Aß peptides on the level of Glu release, as well as how Aß-mediated Glu fluctuation is initiated, remain largely unknown. Here, we fabricated a Glu electrochemical biosensor and in situ quantitatively monitored the release of Glu from a single varicosity of Aß1-42-insulted hippocampal neurons. We found that before the depletion of Glu after 300 min of treatment with Aß1-42, a short-duration (30 min) incubation with Aß1-42 caused a dramatic increase in vesicular Glu release compared to that of a control. Further investigation demonstrated that the density of vesicular glutamate transporter 1 (VGLUT1), which is responsible for transport of Glu into synaptic vesicles, also displayed a significant elevation and then dramatic depletion with the extension of the time of treatment with Aß1-42. These results indicate that at the early stage of AD, Aß1-42 induces excessive Glu release, which may overstimulate the N-methyl-d-aspartic acid (NMDA) receptor, resulting in excitotoxicity and damage to neurons. In this work, the amount of Glu released together with its fluctuations under Aß1-42 oligomers toxicity conditions was monitored for the first time, and such monitoring could provide direct and new insights for current research on Aß1-42-induced abnormalities in neurotransmitter release and neuron functions.

Real-Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core-Shell Nanowire Electrodes.

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living-cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC-core-shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.

The effects of composite photosynthetic bacterial inoculant PS21 on the biochemical characteristics of wheat seedlings under tetrabromobisphenol A stress.

The aim of this study was to investigate whether the composite photosynthetic bacterial inoculant PS21 alleviate the damage inflicted on wheat seedlings by tetrabromobisphenol A (TBBPA). The biochemical characteristics of wheat seedlings were analysed through laboratory simulation after co-treating seedlings with PS21 and 0, 5, 10, 20, 40, 80 and 100 mg kg-1 TBBPA, respectively. The results showed that TBBPA reduced the total chlorophyll content and increased the malondialdehyde (MDA) content. TBBPA increased the soluble sugar content, soluble protein content and activate superoxide dismutase (SOD; EC: 1.15.1.1), catalase (CAT; EC: 1.11.1.6) and peroxidase (POD; EC: 1.11.1.7) at low concentrations, while it reduced soluble sugar content, soluble protein content and decreased the activities of SOD, CAT and POD at high concentrations. At the concentration of 107 CFU mL-1, PS21 could markedly relieve the toxicity of different concentrations of TBBPA on wheat seedlings. Wheat seedlings treated with both TBBPA and PS21 showed a higher soluble sugar content, higher soluble protein content, higher SOD, CAT and POD activities, and a lower MDA content as compared to those treated only with TBBPA. The composite photosynthetic bacterial inoculant PS21 significantly alleviates the damage inflicted on wheat seedlings by TBBPA.

Nanoelectrochemistry reveals how presynaptic neurons regulate vesicle release to sustain synaptic plasticity under repetitive stimuli.

Synaptic plasticity is the ability of synapses to modulate synaptic strength in response to dynamic changes within, as well as environmental changes. Although there is a considerable body of knowledge on protein expression and receptor migration in different categories of synaptic plasticity, the contribution and impact of presynaptic vesicle release and neurotransmitter levels towards plasticity remain largely unclear. Herein, nanoelectrochemistry using carbon fiber nanoelectrodes with excellent spatio-temporal resolution was applied for real-time monitoring of presynaptic vesicle release of dopamine inside single synapses of dopaminergic neurons, and exocytotic variations in quantity and kinetics under repetitive electrical stimuli. We found that the presynaptic terminal tends to maintain synaptic strength by rapidly recruiting vesicles, changing the dynamics of exocytosis, and maintaining sufficient neurotransmitter release in following stimuli. Except for small clear synaptic vesicles, dense core vesicles are involved in exocytosis to sustain the neurotransmitter level in later periods of repetitive stimuli. These data indicate that vesicles use a potential regulatory mechanism to establish short-term plasticity, and provide new directions for exploring the synaptic mechanisms in connection and plasticity.

Nanosensor detection of reactive oxygen and nitrogen species leakage in frustrated phagocytosis of nanofibres.

Exposure to widely used inert fibrous nanomaterials (for example, glass fibres or carbon nanotubes) may result in asbestos-like lung pathologies, becoming an important environmental and health concern. However, the origin of the pathogenesis of such fibres has not yet been clearly established. Here we report an electrochemical nanosensor that is used to monitor and quantitatively characterize the flux and dynamics of reactive species release during the frustrated phagocytosis of glass nanofibres by single macrophages. We show the existence of an intense prolonged release of reactive oxygen and nitrogen species by single macrophages near their phagocytic cups. This continued massive leakage of reactive oxygen and nitrogen species damages peripheral cells and eventually translates into chronic inflammation and lung injury, as seen during in vitro co-culture and in vivo experiments.

(6,6-Dimethyl-1-phenyl-6,7-di-hydro-5H-pyrrolizin-2-yl)(thio-phen-2-yl)methanone.

In the title compound, C20H19NOS, the pyrrolizine ring is essentially planar (r.m.s. deviation = 0.001 Å) while the fused dihydro-pyrrolizine ring adopts an envelope comformation with the C atom bearing the methyl substituents as the flap. The dihedral angles between the pyrrolizine and the phenyl and thio-phene rings are 34.54 (7) and 44.93 (7)°, respectively. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into infinite zigzag chains parallel to the b-axis direction.

Ethyl 2-[6-(4-methyl-benzo-yl)-7-phenyl-2,3-di-hydro-1H-pyrrolizin-5-yl]-2-oxo-acetate.

In the title compound, C25H23NO4, the pyrrolizine ring is approximately planar with an r.m.s deviation from planarity of 0.0053 Å, while the fused di-hydro-pyrrolizine ring adopts an envelope conformation with the C atom connected to two CH2 as the flap. The dihedral angles between the fused ring system and the phenyl and methyl-benzoyl rings are 41.65 (11) and 66.30 (8)°, respectively. In the crystal, weak C-H⋯O hydrogen bonds and C-H⋯π inter-actions occur. One mol-ecule is linked to five adjacent ones through eight hydrogen bonds, forming a three-dimensional network.

Methyl 3-[(chloro-meth-oxy)carbon-yloxy]-7-hy-droxy-cholan-24-oate.

The title compound, C27H43ClO6, is a derivative of urso-deoxy-cholic acid, in which the OH group at the 3-position is substituted by a chloro-meth-oxy-carbon-yloxy substituent and the carb-oxy-lic acid group at the 24-position is methyl-ated. The A and B rings are cis-fused, while all other rings are trans-fused. In the crystal, two adjacent mol-ecules located along the b-axis direction are inter-locked head-to-tail due to weak C-H⋯O hydrogen bonds. Therefore each mol-ecule is linked to four neighbouring mol-ecules by four C-H⋯O hydrogen bonds, with the OH group at the 7-position and the carbonyl O atom of the ester group acting as the acceptor sites.

Chloroplastic photoprotective strategies differ between bundle sheath and mesophyll cells in maize (Zea mays L.) Under drought.

Bundle sheath cells play a crucial role in photosynthesis in C4 plants, but the structure and function of photosystem II (PSII) in these cells is still controversial. Photoprotective roles of bundle sheath chloroplasts at the occurrence of environmental stresses have not been investigated so far. Non-photochemical quenching (NPQ) of chlorophyll a fluorescence is the photoprotective mechanism that responds to a changing energy balance in chloroplasts. In the present study, we found a much higher NPQ in bundle sheath chloroplasts than in mesophyll chloroplasts under a drought stress. This change was accompanied by a more rapid dephosphorylation of light-harvesting complex II (LHCII) subunits and a greater increase in PSII subunit S (PsbS) protein abundance than in mesophyll cell chloroplasts. Histochemical staining of reactive oxygen species (ROS) suggested that the high NPQ may be one of the main reasons for the lower accumulation of ROS in bundle sheath chloroplasts. This may maintain the stable functioning of bundle sheath cells under drought condition. These results indicate that the superior capacity for dissipation of excitation energy in bundle sheath chloroplasts may be an environmental adaptation unique to C4 plants.

Dosimetric evaluation of helical tomotherapy and volumetric-modulated arc therapy for malignant pleural mesothelioma: a planning study for dose escalation.

BACKGROUND: To compare the dosimetric differences between helical tomotherapy (HT) and volumetric-modulated arc therapy (VMAT) treatment plans for inoperable malignant pleural mesothelioma (MPM). METHODS: Ten patients with inoperable MPM were retrospectively planned with the HT and VMAT techniques, and the dose-volume histogram (DVH)-based parameters of the planning target volume (PTV) and organs at risk (OARs) were compared. RESULTS: Compared with the VMAT plans, the target homogeneity index (HI) and conformity index (CI) of the HT plans were significantly better (HI: 1.04±0.01 vs. 1.11±0.03, CI: 0.80±0.07 vs. 0.71±0.12, respectively) (P<0.001, P=0.013, respectively). Regarding the OARs, including the ipsilateral lung, contralateral lung, heart, and spinal cord, the differences among the V30 (Vx: fraction of volume receiving >5, 10, 20, and 30 Gy, respectively) of the ipsilateral lung and V5, V10, and V20 of the contralateral lung were statistically significant (P=0.031, P=0.030, P=0.021, P=0.003, respectively). However, there was no significant differences between HT plans and VMAT plans, regarding the V5, V10 and V20 of the ipsilateral lung, V3 of the contralateral lung, V5 and Dmean of the heart, and Dmax of the cord. The treatment delivery time of the VMAT was significantly shorter than that of the HT (mean delivery time: 3.27±1.65 vs. 11.11±3.75 min, respectively) (P<0.001). CONCLUSIONS: Compared to the VMAT plans, the HT plans not only demonstrated more optimal target coverage and conformity but also considerably reduced the dose-volume parameters of the OARs in both low-dose areas in contralateral lung and high-dose areas in ipsilateral lung and contralateral lung, which is correlated to radiation injury. However, the treatment delivery time of the HT plans was longer.

Hesperidin alleviates insulin resistance by improving HG-induced oxidative stress and mitochondrial dysfunction by restoring miR-149.

BACKGROUND: Hesperidin, a natural flavanone, has been proven to have multiple protective effects in diabetic rats, such as antioxidant, anti-inflammatory and anti-apoptotic effects. However, the molecular mechanisms underlying the effects of hesperidin are not well elucidated. METHODS: LO2 cells were stimulated with high glucose (HG, 33 mM) for 24 h to establish a model of oxidative stress. Then, cell viability was determined using the MTT assay. The antioxidant activities, including the reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, mitochondrial membrane potential (MMP) and adenosine-triphosphate (ATP) production, were measured with the corresponding kits. The levels of gene expression, protein expression and methylation were detected using qRT-PCR, western blotting and methylation-specific PCR (MSP) assays, respectively. RESULTS: Compared to the NG treatment, hesperidin treatment increased the viability and improved the oxidative stress, mitochondrial dysfunction and insulin resistance of HG-treated LO2 cells, and these effects were correlated with heightened SOD and GPx activities, increased MMP level and ATP generation, reduced MDA, ROS and glucose levels, and activated GSK3ß/AKT and inactivated IRS1 signals. Mechanistically, hesperidin treatment enhanced the miR-149 expression level by reducing its promoter methylation by inhibiting DNMT1. Importantly, knockdown of miR-149 obviously abolished the biological roles of hesperidin. CONCLUSIONS: Our findings demonstrated that hesperidin treatment ameliorated HG-induced insulin resistance by reducing oxidative stress and mitochondrial dysfunction partly by suppressing DNMT1-mediated miR-149 silencing.

Poly[tetra-µ-cyanido-dipyridine-cadmium(II)zinc(II)].

In the title coordination polymer, [CdZn(CN)(4)(C(5)H(5)N)(2)](n), the Zn(II) atom (site symmetry 222) adopts a distorted ZnC(4) tetra-hedral geometry, being coordinated by four crystallographically equivalent cyanide ions. The cyanide ion bridges to a Cd(II) centre via its N atom. The Cd atom (site symmetry 2/m) coordination is a distorted CdN(6) octa-hedron, arising from four cyanide N atoms and two pyridine N atoms. The complete pyridine mol-ecule is generated by m symmetry, with the N atom and one C atom lying on the reflecting plane. In the crystal, the bridging cyanide ions result in a three-dimensional network.

Preliminary results on anal cancer by applying intensity modulated radiotherapy and synchronous capecitabine chemotherapy simultaneously.

BACKGROUND: Anal cancer is a rare clinical disease with the incidence rate of 1-2/10 million. The present study aims to assess the feasibility, safety and short-term outcome of the simultaneous integrated boost intensity-modulated radiotherapy (SIB-IMRT) schedule with oral capecitabine in patients with anal cancer. METHODS: From September 2009 to February 2014, a total of 10 patients with anal carcinoma were treated with SIB-IMRT in 32 daily fractions of 1.8 Gy to the primary tumor and macroscopically involved lymph nodes and 32 fractions of 1.55 Gy electively to the bilateral iliac and inguinal lymph node areas with concurrent capecitabine 625 mg/m2 twice daily 5 days/week. Two patients received a sequential radiation boost dose of 2×1.8 Gy on macroscopic residual tumor in week 5 of treatment. Acute and late toxicity was assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. RESULTS: All patients completed chemoradiation without any treatment break. Grade 3 acute skin toxicity was observed in 4 patients (40%). No grade 4 toxicity was observed. Mean follow-up was 20 months (range: 6-60 months). The 2-year-local control, colostomy-free survival, distant metastases-free survival and overall survival (OS) rates were 100% (10/10), 90% (9/10), 90% (9/10), and 90% (9/10), respectively. CONCLUSIONS: SIB-IMRT with concomitant capecitabine 625 mg/m2 b.i.d. on irradiation days resulted in an acceptable safety profile, and proved to be a tolerable and effective treatment regimen for locally advanced anal cancer. However, a larger number of patients and a longer follow-up are required to assess its potential superiority.

A simple and effective method to discern the true commercial Chinese cordyceps from counterfeits.

The Chinese cordyceps, a complex of the fungus Ophiocordyceps sinensis and its species-specific host insects, is also called "DongChongXiaCao" in Chinese. Habitat degradation in recent decades and excessive harvesting by humans has intensified its scarcity and increased the prices of natural populations. Some counterfeits are traded as natural Chinese cordyceps for profit, causing confusion in the marketplace. To promote the safe use of Chinese cordyceps and related products, a duplex PCR method for specifically identifying raw Chinese cordyceps and its primary products was successfully established. Chinese cordyceps could be precisely identified by detecting an internal transcribed spacer amplicon from O. sinensis and a cytochrome oxidase c subunit 1 amplicon from the host species, at a limit of detection as low as 32 pg. Eleven commercial samples were purchased and successfully tested to further verify that the developed duplex PCR method could be reliably used to identify Chinese cordyceps. It provides a new simple way to discern true commercial Chinese cordyceps from counterfeits in the marketplace. This is an important step toward achieving an authentication method for this Chinese medicine. The methodology and the developmental strategy can be used to authenticate other traditional Chinese medicinal materials.

Growth and antioxidant responses in Jatropha curcas cotyledons under lead stress.

Jatropha curcas embryos were grown in vitro to observe the effects of lead on cotyledon responses. The cotyledon biomass increased initially and then decreased with increasing lead concentration. The SOD activity increased gradually up to 200 microM and then decreased. The POD activity showed a similar trend. The CAT activity was increased at all lead concentrations, the highest activity being observed at 200 microM. However, the PAL activity was inhibited significantly except for 100 microM. Anaylsis by electrophoresis suggested a significant correlation between lead concentration and patterns of SOD, POD and CAT isoenzymes, and these results were consistent with changes of the antioxidant enzyme activities as assayed in solution.