Abstract  The present study was carried out to determine the plexus sacralis and its branches in sparrowhawk (Accipiter nisus). Fifteen sparrowhawk were used in this study. Following the anaesthesing of the materials, cavity of the body was opened. Animals were fixed with formaldehyde after draining of their bloods. The nerves of plexus sacralis were separately dissected and photographed. Plexus sacralis was formed by the union of the branches of the synsacral spinal nerves which leave from ventrolaterale of os lumbosacrale. The plexus consisted of six (4-9) synsacral spinal nerves in sparrowhawk (Accipiter nisus). The nerves originating from plexus sacralis from cranial to caudal were nervus coxalis caudalis, the common root of nervus peroneus and nervus tibialis, nervus cutaneus femoris caudalis and the common branches of rami musculares. It was determined that general macroanatomical shapes of plexus sacralis and the distribution of nerves originating from this plexus were found to be similar with the other bird species in hawk which is one of the predatory bird species. However, it was determined that responsible for innervation of the leg and foot nervus peroneus and nervus tibialis common root and terminal branches of these nerves were significantly thick and strong in appearance.

Abstract  C-f/SiBOC was fabricated from 2D carbon fabric as reinforcement and slurry-containing boron-modified phenol formaldehyde (BPF) resin with silicon as matrix resin using reaction-bonded silicon carbide method. The processing involves synthesis of (BPF) resin by reacting various amount of boric acid with phenol formaldehyde resin, polymer to ceramic transformation at 1450 degrees C under argon atmosphere, with and without silicon, thermal transformation of the polymer matrix composite into a ceramic matrix composite and evaluation of isothermal oxidation for ceramics and its composites at 1000, 1250 and 1500 degrees C. The ceramic studies, confirmed the formation of B4C, SiC and SiB4 (SiBOC) mixed phase and the role of boron as a catalyst for graphitisation of free carbon present in the ceramic. Oxidation of C-f/SiBOC composite at various temperatures leads to the formation of borosilicate glass which heals the cracks, hindering the inwards diffusion of oxygen.

Abstract  In this work, a free template method is used to synthesize nanostructured analcime (ANA) zeolite from natural source and the prepared zeolite is applied as substrate to fabricate a modified electrode. To this aim, ANA zeolite nanoparticles are synthesized by hydrothermal method using amorphous silica extracted from stem of sorghum ash (SSA) with approximately 92.34% purity in the absence of organic template. The effects of different parameters such as Si/Al ratio, heating time, temperature and stirring time on synthesis and crystallinity of zeolite are optimized. The synthesized zeolite is characterized using Xray diffraction, Fourier-transform infrared and scanning electron microscopy (SEM) techniques. SEM shows that ANA zeolite nanoparticles appear as spherical particles contains some smaller nanoparticles which arranged in regular pattern to each other. Also, some fibers consist of nanoparticles in range of 30-48 nm are observed.

The modified carbon paste electrode (CPE) is prepared based on ANA modified with Ni species (Ni/ANACPE). The electrochemical oxidation of formaldehyde is studied at Ni/ANACPE in alkaline solution. From cyclic voltammetry studies, it is shown that Ni/ANACPE can facilitate the oxidation of formaldehyde compared to CPE. The effects of some parameters such as scan rate of potential, concentration of formaldehyde, amount of Ni-zeolite are investigated on the oxidation of formaldehyde. Also using chronoamperometry technique, the catalytic rate constant (k) for oxidation of formaldehyde is found to be 7.56 x 10(4) cm(3) mol(-1) s(-1). (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Abstract  The biosynthesis of silver nanoparticles (Ag-NPs) using the hydrosol extract of the dry stem bark of Acacia mearnsii as reducing and capping agents, and their antinociceptive properties are hereby reported. By varying the temperature and reaction time, the temporal evolution of the optical and morphological properties of the as-synthesized material was investigated. The NPs were characterized by UV-visible absorption spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDS) and x-ray diffractometry (XRD) The optical analyses show that the position of the maximum surface plasmon resonance (SPR) peak is red-shifted as the reaction temperature decreased. The TEM micrographs show that the as-synthesized Ag-NPs are spherical while the X-ray diffraction shows that the material is highly crystalline with face-centered cubic structures. The anti-inflammatory efficacy, analyzed by the formalin model, indicates that the as-synthesized Ag-NPs are very effective, with an inhibition rate of about 76%.

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Abstract  Male albino Wistar rats were exposed to 10 nm gold nanoparticles at a dose of 2000 mu g/kg together with or without propolis (50 mg/kg) for 15 consecutive days. Fresh renal biopsies from of all investigated rats were cut rapidly, fixed in neutral buffered formalin, subjected to histological processing, and examined for microanatomical alterations. Propolis gave full protection against glomerular congestion and renal tubule hyaline casts. It demonstrated partial amelioration against glomerular capillary dilatation, tubular cloudy swelling, necrosis, and degeneration together with interstitial blood capillaries dilatation and haemorrhage induced by nanoparticle toxicity. On the other hand, propolis showed no protective effect against renal cells pyknosis, karyolysis, apoptosis and renal hydropic degeneration together with collecting tubules atrophy and degeneration induced by the nanoparticles. Thus propolis can augment the antioxidant defence against the severity of some alterations in the renal tissues. This ameliorative role might be related to the antioxidants content of propolis that protect the renal tissues from free radicals and oxidative stress.

Abstract  Mannheimia haemolytica is responsible for considerable economic losses to sheep, goats, and cattle and other livestock industries in Egypt. This study aimed to evaluate the effectiveness of a newly developed gamma irradiation vaccine against Mannheimia haemolytica in comparison to a formalin-killed vaccine. Three groups of rabbits were used in this study. Group 1 animals were inoculated with 4 x 10(9) bacterial cells per dose of the formalin-killed vaccine. Group 2 was inoculated with 2 x 109 bacterial cells per dose of gamma-irradiated vaccine. Group 3 (control group) was injected with 2 mL of sterile PBS. The vaccines were injected subcutaneously into experimental animals twice with 3-week intervals between inoculations. Three weeks after the second vaccination dose, the animals in all groups were infected with M. haemolytica twice with 1-week intervals between inoculations. Blood samples were collected weekly after the first vaccination until one week after the second M. haemolytica infection challenge. ELISA results revealed that the gamma irradiation vaccine developed in this study provided protective effects that reached high levels at the time of challenge. Furthermore, the second dose of gamma irradiation vaccine could act as a booster dose resulting in increased antibody production.

Abstract  In the recent years, organic electrode materials have attracted tremendous attention and becoming promising electrode candidates for the green and sustainable lithium-ion batteries. A novel hypercrosslinked Poly-Pillar[5]quinone (Poly-P5Q) polymer was prepared and applied as electrode material in Li-ion batteries. Poly-P5Q is the oxidized form of Poly-Dimethoxypillar[5]arene (Poly-DMP5A) which was obtained from the condensation of dimethoxypillar[5]arene and formaldehyde dimethyl acetal using Friedel-Crafts reaction. The prepared materials were characterized by C-13 solid state NMR, FTIR, SEM, EDX and TGA analysis. The Poly-P5Q cathode showed an initial discharge-capacity up to 105 mAh g(-1) whereas it retained 82.3% of its initial discharge capacity after 100 charge-discharge cycles at a current speed of 100 mA g(-1) in the potential window between 1.75 to 3.25 V. In future, research in this direction will provide great insight for the development of novel polymers from various small organic molecules as a stable and high performance electrode materials for green lithium-ion batteries. (C) 2017 Elsevier Ltd. All rights reserved.

Abstract  Hydrogen sulfide (H2S) is an endogenous neuromodulator produced mainly by the enzyme cystathionine gamma-lyase (CSE) in peripheral tissues. A pronociceptive role of endogenously produced H2S has been previously reported by our group in a model of orofacial inflammatory pain. Using the established persistent orofacial pain rat model induced by complete Freund's adjuvant (CFA) injection into temporomandibular joint (TMJ), we have now investigated the putative role of endogenous H2S modulating hypernociceptive responses. Additionally, plasmatic extravasation on TMJ was measured following different treatments by Evans blue dye quantification. Thus, rats were submitted to Von Frey and Formalin tests in orofacial region before and after pharmacological inhibition of the CSE-H2S system combined or not with CFA-induced TMJ inflammation. Pretreatment with CSE inhibitor, propargylglycine (PAG; 88.4 μmol/kg) reduced temporomandibular inflammatory pain when injected locally as well as systemically. In particular, local PAG injection seems to be more effective for hypernociceptive responses in orofacial persistent inflammation since its action is evidenced in the majority analyzed periods of the inflammatory process compared to its systemic use. Moreover, local injection seems to act on temporomandibular vascular permeability, evidenced by decreased plasmatic extravasation induced by local PAG administration. Our data are consistent with the notion that the endogenous synthetized gas H2S modulates persistent orofacial pain responses revealing the pharmacological importance of the CSE inhibitor as a possible therapeutic target for their control.

Abstract  OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP(ND251)) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP(ND251), as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.

Abstract  Traditionally Berberis species have been used as anti-inflammatory, anti-rheumatic, analgesic and anti-anemic drugs. This study was aimed to determine chemical constituents and to assess analgesic, anti-inflammatory and hematological effects of the crude extract of the berries of Berberis baluchistanica to verify these folkloric claims. Phytochemical screening, carried out by using different chemical reagents and techniques like Thin Layer Chromatography (TLC) and Fourier Transform infra-Red (FTIR) indicated presence of flavonoids, saponins, phytosterols and carbohydrates including reducing sugars. Analgesic and anti-inflammatory activities were assessed on mice by using acetic acid induced writhing method and formalin method. Potent anti-inflammatory and analgesic effects were observed during these experiments. The extract also showed anti anemic effect as it increased the levels of hemoglobin and red blood cells significantly. Increase in the platelet count was also noted. The extract of the berries was used at oral doses of 300 and 500 mg/kg during experiments. Anti-inflammatory and analgesic activities were determined by comparing with the standard i.e. aspirin 300 mg/kg. Both doses produced significant anti-inflammatory and analgesic activities at P<0.05. These activities were seemingly attributable to flavonoid and saponin contents of the drug. These results justify the folkloric claims that the drug could be used as good anti-inflammatory, antirehumatic, analgesic and anti-anemic drug. However, further chemical investigations on the drug are suggested for isolation and identification of compounds that could be safer and more effective than the currently available medicines in treating these disorders.

Abstract  INTRODUCTION: This clinical study was conducted to measure the endotoxin levels in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs). In addition, the effectiveness of RC procedures in reducing the endotoxin levels in RCs was monitored.

METHODS: Paired samples of infected RCs and exudates from AAAs were collected from 10 subjects by using paper points. RCs samples were collected before (RCS1) and after chemomechanical preparation (CMP) (RCS2), after 17% EDTA (RCS3), and after 30 days of intracanal medication (Ca[OH]2 + chlorhexidine) (RCS4). A turbidimetric kinetic limulus amebocyte lysate assay was used for the measurement of endotoxins.

RESULTS: Endotoxins were detected in 100% of the baseline samples of AAAs and RCs (RCS1) with median values of 175 EU/mL and 41.5 EU/mL, respectively (P < .05). After CMP (RCS2), endotoxins were reduced to a median value of 0.54 EU/mL (P < .05). Subsequent irrigation with EDTA (RCS3) failed to present a significant effectiveness in reducing the endotoxin levels (median= 0.37 EU/mL) (P = .07). However, intracanal medication for 30 days (RCS4) reduced endotoxins to median values of 0.03 EU/mL (P < .01).

CONCLUSIONS: The present study revealed a strong association between the high levels of endotoxins found in AAAs and RCs collected from the same tooth. Moreover, the effectiveness of CMP in reducing the endotoxin levels from RCs in acute endodontic infection was improved by the use of RC medication.

Abstract  Geraniol (GER) is a monoterpene alcohol with various biochemical and pharmacological properties present in the essential oil of more than 160 species of herbs (especially the Cymbopogon genus). In this study, we evaluated the antinociceptive activity of GER in behavioural and electrophysiological in vitro experimental models of nociception using male Swiss mice. GER (12.5, 25 or 50 mg/kg i.p. and 50 or 200 mg/kg p.o.) reduced the number of writhes induced by acetic acid. The opioid antagonist naloxone (5 mg/kg s.c.) administered in mice subsequently treated with GER (25 mg/kg i.p.) did not reverse such antinociceptive activity, suggesting a non-opioid pathway for the mechanism of action. GER (12.5, 25 and 50 mg/kg i.p.) reduced paw licking time in the second phase of the formalin test. Also, in the glutamate test, GER when administered 50 mg/kg i.p. reduced paw licking time, probably modulating glutamatergic neurotransmission. GER blocked reversibly components of the compound action potential (CAP) recorded in isolated sciatic nerve in a concentration- and drug exposure time-dependent manner: 1 mM to 120 min. for the first component and 0.6 mM to 90 min. for the second component. The IC50 was calculated for the peak-to-peak amplitude (PPA) at 0.48 ± 0.04 mM. The conduction velocity was also reduced by exposure to GER starting from the concentration of 0.3 mM for both components of the CAP. In conclusion, it is suggested that GER has antinociceptive activity, especially in pain related to inflammation, and in part related to reduced peripheral nerve excitability.

Abstract  BACKGROUND: Ketamine is a controlled substance and often illegally used as a recreational drug primarily by young adults. Increasing ketamine abusers associated with lower urinary tract symptoms have been reported at hospitals in recent years. Here we used a murine model to explore the changes of bladder in order to elucidate its pathogenesis.

METHODS: ICR mice were randomly distributed into control and ketamine groups and received daily intraperitoneal injection of saline and ketamine (30 mg/kg), respectively. The bladders were excised and processed for histology at 4, 8 and 12 weeks. Tryptase and E-cadherin were investigated by immunohistochemistry in bladder tissues from ketamine-treated and control mice to assess the mast cell activation and junction protein expression.

RESULTS: After ketamine treatment, the bladder changed to be hyperemic, inflamed, and with more fissures in mucosa. Compared with control group, the number of tryptase-positive mast cells significantly increased, which was 6.98 ± 2.89 and 23.00 ± 6.48 cells per field (100×) at 8 and 12 weeks, respectively (P = 0.016 and P = 0.003, respectively). Additionally, the expression of E-cadherin in ketamine-treated mice bladder tissue was significantly lower than that in the control tissues, P < 0.001.

CONCLUSIONS: Increased mast cells in bladder wall and downregulated expression of E-cadherin junction protein in epithelial cells were probably associated with interstitial inflammation and fissures in mucosa. It implied that ketamine induced an interstitial cystitis.

Abstract  UNLABELLED: Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 μM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible.

IMPORTANCE: The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements.

Abstract  Late endosomes are a major trafficking hub in the cell at the crossroads between endocytosis, autophagy, and degradation in lysosomes. Herein is disclosed the first small molecule allowing their selective imaging and monitoring in the form of a diazaoxatriangulene fluorophore, 1a (hexadecyl side chain). The compound is prepared in three steps from a simple carbenium precursor. In nanospheres, this pH-sensitive (pKa = 7.3), photochemically stable dye fluoresces in the red part of visible light (601 and 578 nm, acid and basic forms, respectively) with a quantum yield between 14 and 16% and an excited-state lifetime of 7.7-7.8 ns. Importantly, the protonated form 1a·H(+) provokes a specific staining of late endosome compartments (pH 5.0-5.5) after 5 h of incubation with HeLa cells. Not surprisingly, this late endosome marking depends on the intra-organelle pH, and changing the nature of the lipophilic chain provokes a loss of selectivity. Interestingly, fixation of the fluorophore is readily achieved with paraformaldehyde, giving the possibility to image both live and fixed cells.

Abstract  Multicomponent reactions (MCRs) represent an ideal organic synthesis tool for the rapid construction of complex molecules due to their step and atom economy. Compared to two-component reactions, the development of new MCRs has been greatly limited during the 170 years since the first MCR was reported. Theoretically, the trapping of an active intermediate generated from two components by a third component could change the traditional two-component reaction pathway, leading to the discovery of MCRs. We report an example of the trapping of α-imino enols generated in situ from 1-sulfonyl-1,2,3-triazoles via α-imino metal carbene species by vinylimine ions using C(2)-substituted indoles and paraformaldehyde as precursors in the presence of a rhodium(II) catalyst. The traditional enol-ketone transformation pathway was suspended by the trapping procedure and efficiently switched to an MCR pathway to produce α-amino-β-indole ketones in moderate to good yields. Unexpectedly, the resulting products and the theoretical density functional theory (DFT) calculation results indicated that the enolic carbon had a stronger nucleophilicity than the well-known traditional enamic carbon in the trapping process. The reaction mechanism was investigated using control experiments and detailed DFT calculations, and the synthetic application of the products was also illustrated. The developed strategy provides a mild and rapid access to α-amino-β-indole ketones and suggests a rationale for the discovery of MCRs by trapping an active intermediate with a third component in a traditional two-component reaction pathway.

Abstract  The purpose of this study was to investigate the neurokinin receptor-1 (NKR-1) protein expression in ocular tissues before and after supra-cataract threshold ultraviolet radiation (UVR-B peak at 312 nm) exposure in vivo in a mouse model. Six-week-old C57Bl/6 mice were unilaterally exposed to a single (2.9 kJ/m2) and an above 3-fold UVR-B cataract threshold dose (9.4 kJ/m2) of UVR. UVR-exposure (λpeak = 312 nm) was performed in mydriasis using a Bio-Spectra exposure system. After latency periods of 3 and 7 days, eyes were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with fluorescence coupled antibody for NKR-1 and DAPI for cell nuclei staining. Control animals received only anesthesia but no UVR-exposure. Cataract development was documented with a Leica dark-field microscope and quantified as integrated optical density (IOD). NKR-1 is ubiquitously present in ocular tissues. An above 3-fold cataract threshold dose of UV-radiation induced NKR-1 upregulation after days 3 and 7 in the epithelium and endothelium of the cornea, the endothelial cells of the iris vessels, the pigmented epithelium/stroma of the ciliary body, the lens epithelium, pronounced in the nuclear bow region and the inner plexiform layer of the retina. A significant upregulation of NKR-1 could not be provoked with a single cataract threshold dose (2.9 kJ/m2 UVR-B) ultraviolet irradiation. All exposed eyes developed anterior subcapsular cataracts. Neurokinin-1 receptor is present ubiquitously in ocular tissues including the lens epithelium and the nuclear bow region of the lens. UV-radiation exposure to an above 3-fold UVR-B cataract threshold dose triggers NKR-1 upregulation in the eye in vivo. The involvement of inflammation in ultraviolet radiation induced cataract and the role of neuroinflammatory peptides such as substance P and its receptor, NKR-1, might have been underestimated to date.

Abstract  Colleters are secretory structure present on many families including Rubiaceae. Particular characteristics have been described about colleters secretory cells, however senescence process are still under debate. Tocoyena bullata (Vell.) Mart. (Rubiaceae) shoot apex were collected at Jardim Botânico do Rio de Janeiro, RJ/Brazil. Stipules were separated and fragments were fixed in 2.5% glutaraldehyde and 4.0% formaldehyde in 0.05 m sodium cacodylate buffer, pH 7.2, post fixed in 1.0% osmium tetroxide in the same buffer, dehydrated in acetone, critical-point-drying, sputtered coated and observed. For light microscopy fragments were fixed and dehydrated, infiltrated with historesin and stained with 1% toluidine blue. For transmission electron microscopy, the samples were infiltrated with Epoxi resin. Colleters are present on stipule adaxial surface. On the beginning of development, these structures are recognized as small projections. Later on, colleters differentiated and secrete by cuticle rupture. The colleters senescence occurs in a concomitant and indissoluble way of programmed cell death. Ultrastructural analyses during the process strongly suggest the senescence is based on a non-autolitic programmed cell death. T. bullata colleters, present at stipule abaxial surface are cylindrical secretory structures. Colleters secretory cells originated as stipule projections; differentiate; secrete and senesce by programmed cell death. The secretion and the cell dead occurs in a concomitantly and indissoluble way.

Abstract  Objective To prepare the monoclonal antibody (mAb) against influenza A virus (H1N1) using purified viral particles as antigen and investigate the characterization of host cells infected with influenza virus utilizing the mAb. Methods A/PR/8 (H1N1) virus was cultured in embryonated chicken eggs and further purified by differential and density gradient centrifugation. The structure of viral particles was identified by transmission electron microcopy (TEM). Immunogenicity of purified virus was evaluated by Balb/c mice immunized with formalin-inactivated virus. Hybridomas secreting mAbs were established through a fusion of Sp2/0 myeloma cells and splenocytes from the mice immunized with the virus. The characteristics of mAb were identified by ELISA, immunofluorescence assay (IFA), Western blotting, hemagglutinin inhibition assay (HI) and microneutralization assay. The outside hemagglutinin (HA) on the plasma membrane of the host MDCK cells in which the viruses were propagated and the apoptosis of MDCK cells infected with the viruses were measured using flow cytometry. Cell-based ELISA was established using mAb specific to HA. Subsequently, the growth of virus was analyzed by cell-based ELISA. Results Transmission electron microscopy revealed that the physical structures of the purified virus were spherical, elliptical and extended threadlike. Serum IgG titer to influenza virus showed a progressive increase, and the IgG titer reached 106 after the immunization for 6 weeks. Six hybridoma clones secreting mAb specific to A/PR/8 were developed by hybridoma technology. The HI and neutralization activities of PR8-10 mAb were significantly higher than those of the other mAbs. HI and neutralization titers of PR8-10 mAb were 1:2048 and 1:640, respectively. IFA and Western blotting confirmed that PR8-10 mAb could recognize HA. Flow cytometry showed that PR8-10 mAb also recognized HA on the membrane of MDCK in which the viruses were replicated and virus infection induced the apoptosis of MDCK cells. Based on the previous test results that PR8-10 mAb was able to recognize HA on the membrane of the host cells in which the viruses were replicated, cell-based ELISA we established was good at analyzing the growth of virus in MDCK cells. Conclusion We obtained whole viral particles that were demonstrated to be able to stimulate the production of a high IgG titer in a mouse model with formalin-inactivated viral particles, and successfully prepared the mAb against H1N1 of high binding affinity and neutralization potency. HA-specific mAb can be used to analyze the characteristics of virus infection process and the effect of virus infection on the host cells as well.

Abstract  Methods to detect protozoa in water samples are expensive and laborious. We evaluated the formalin/ether concentration method to detect Giardia sp., Cryptosporidium sp. and Toxoplasma in water. In order to test the properties of the method, we spiked water samples with different amounts of each protozoa (0, 10 and 50 cysts or oocysts) in a volume of 10 L of water. Immunofluorescence assay was used for detection of Giardia and Cryptosporidium. Toxoplasma oocysts were identified by morphology. The mean percent of recovery in 10 repetitions of the entire method, in 10 samples spiked with ten parasites and read by three different observers, were for Cryptosporidium 71.3 ± 12, for Giardia 63 ± 10 and for Toxoplasma 91.6 ± 9 and the relative standard deviation of the method was of 17.5, 17.2 and 9.8, respectively. Intraobserver variation as measured by intraclass correlation coefficient, was fair for Toxoplasma, moderate for Cryptosporidium and almost perfect for Giardia. The method was then applied in 77 samples of raw and drinkable water in three different plant of water treatment. Cryptosporidium was found in 28 of 77 samples (36%) and Giardia in 31 of 77 samples (40%). Theses results identified significant differences in treatment process to reduce the presence of Giardia and Cryptosporidium. In conclusion, the formalin ether method to concentrate protozoa in water is a new alternative for low resources countries, where is urgently need to monitor and follow the presence of theses protozoa in drinkable water.

Abstract  AIMS: To investigate whether the protective effect of testosterone on the development of temporomandibular joint (TMJ) nociception in male rats is mediated by the activation of central opioid mechanisms.

METHODS: Experiments were performed on 156 male Wistar rats. A pharmacologic approach was used to assess the ability of opioid receptor antagonists infused into the dorsal portion of the brainstem and adjacent to the caudal component (subnucleus caudalis) of the spinal trigeminal nucleus to block the protective effect of testosterone in male rats. The TMJ injection of 0.5% formalin was used as a nociceptive stimulus. One-way or two-way ANOVA was used for data analyses.

RESULTS: The injection of 0.5% formalin into the TMJ induced a significant nociceptive behavior in gonadectomized male rats (P < .05), but not in naïve, sham, and testosterone-replaced gonadectomized rats, confirming that testosterone prevents the development of TMJ nociception. The injection of either the nonselective opioid receptor antagonist naloxone (15 μg) or the simultaneous injection of the μ-opioid receptor antagonist Cys2, Tyr3, Orn5, Pen7amide (CTOP, 30 μg) and the κ-opioid receptor antagonist Nor-Binaltorphimine (Nor-BNI, 90 μg) significantly increased the 0.5% formalin-induced behavioral response in sham and testosterone-replaced gonadectomized rats (P < .05) but had no effect in gonadectomized rats. However, the injection of each selective opioid receptor antagonist alone or the simultaneous injection of μ- or κ- and δ-opioid receptor antagonists had no effect.

CONCLUSION: These findings indicate that the protective effect of endogenous testosterone on the development of TMJ nociception in male rats is mediated by the activation of central opioid mechanisms. Furthermore, the coactivation of central μ- and κ-opioid receptors is necessary for testosterone to protect male rats from developing TMJ nociception.

Abstract  The adsorption of the hydrogen peroxide (H2O2) molecule, which is known as the common form of reactive oxygen species in living cells, was investigated theoretically over pure graphene and heteroatom- (nitrogen-, boron-, and sulfur-) and metal-atom- (silver-, gold-, copper-, palladium-, and platinum-) doped graphene surfaces using the density functional theory (DFT) method. This study involved the optimization of pure and doped graphene surfaces, adsorption of the gas molecule on top of the doped atoms and neighboring carbon atoms, and analysis of the behavior of the gas molecule over the various adsorption sites. First principles calculations revealed that the copper-doped and silver-doped graphene surfaces are the most thermodynamically favorable surfaces for the direct formation of water molecules. Moreover, the sulfur-doped surface exhibits a superior performance among the heteroatom-doped surfaces. Additionally, the gap between the orbital energies of the system was found to have an effect on the surface behavior of the H2O2 molecule.

Abstract  Study of the mode of action (MOA) relating exposure to a given chemical with an associated adverse outcome is an iterative process with each iteration driven by new understandings of the relevant biology. Here, we revisit a previously described, MOA-based clonal growth model of the human respiratory tract cancer risk associated with formaldehyde inhalation. Changes reflect a better understanding of populations of cells at risk of carcinogenic transformation in the pharynx, larynx and respiratory bronchiolar portions of the human respiratory tract and inclusion of basal cells in the pool of cells at risk. The focus of this report is not on cancer risk per se, but rather on the sensitivity of model parameters and predicted risks to alternative descriptions of the fraction of cells at risk for carcinogenic transformation. For a population of formaldehyde-exposed nonsmokers, revised specification of cells at risk resulted in changes in both parameter estimates and in predicted risks. Compared to our previous assessment, predicted additional risks were up to 87% greater at exposure levels ≤1 ppm, but up to about 130% lower at high exposure levels (2-5 ppm). While this work should not be considered an update to MOA-based risk assessments for formaldehyde described previously, it illustrates the sensitivity of parameter estimates and risk predictions to the quantitative specification of cells at risk of carcinogenic transformation and, therefore, the motivation for describing the relevant biology as accurately as possible.

Abstract  The present study is the first to show overall prevalences of intestinal parasites among breeding kennel dogs in Japan. A total of 573 fresh fecal samples were collected from dogs at 12 breeding kennels. Giardia-specific coproantigen was examined by ELISA kit (SNAP(®) Giardia, IDEXX Laboratories, Inc., Maine, USA). Other intestinal parasites were determined microscopically using the formalin-ethyl acetate sedimentation technique. Overall prevalences of two genera of protists, Giardia spp. and Cystoisospora spp., were 25.7 and 1.2 %, respectively. The prevalence of helminthes was recorded as: Toxocara canis 0.2 %, Toxascaris leonina 0.9 %, Ancylostoma caninum 0.2 %, Trichuris vulpis 2.1 %, and Spirometra erinacei 0.4 %. According to age categories, Giardia spp., Cystoisospora spp., and T. leonina in <1-year-old dogs were significantly more prevalent than in ≥ 1-year-old dogs (61.0 vs. 19.8 %, P < 0.0001; 7.3 vs. 0.2 %, P < 0.0001; and 4.9 vs. 0.2 %, P < 0.001; respectively). With respect to fecal condition, the prevalences of T. leonina and T. vulpis were significantly higher in unformed stool dogs than in formed ones (2.4 vs. 0 %, P < 0.01, and 4.3 vs. 0.8 %, P < 0.05, respectively). In all of the breeding kennels except for one kennel, intestinal parasite infections were found at the high prevalent, ranging from 16.0 to 70.0 %.

Abstract  BACKGROUND: Inappropriate platelet activation is known to be associated with various thrombotic disorders. Platelet-monocyte aggregates (PMAs), whose formation is mediated by platelet surface P-selectin (CD62P), can be used as a reliable marker to detect platelet activation. Previous studies have generally detected PMAs through flow cytometry-based approaches. Recently, the ADAM(®) image cytometer (Nanoentek Inc., Seoul, Korea) was developed for image-based cellular analysis. In this study, we detected PMAs with the ADAM(®) cytometer, evaluated the reproducibility of the measurements made by the ADAM(®) cytometer, and compared the abilities of the ADAM(®) cytometer and a flow cytometric assay to detect PMAs.

METHODS: Whole blood samples were collected from patients. Within 5 minutes of collection, anticoagulated whole blood samples were fixed in 10% paraformaldehyde and 5% glyoxal. Nineteen clinical specimens were collected; each was analyzed three times with the ADAM(®) cytometer in order to assess the reproducibility of its measurements. To compare the ability of the ADAM(®) cytometer with that of a flow cytometer to detect PMAs, each cytometer was used for 23 clinical samples and the correlation of the measurements was determined.

RESULTS: The PMA measurements made by the ADAM(®) cytometer showed good reproducibility (CV < 10% for all specimens). Moreover, the PMA measurements made by the ADAM(®) cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944).

CONCLUSIONS: The ADAM(®) cytometer is a suitable alternative method to the flow cytometry-based assays. Since the ADAM cytometer does not need specialized instrument knowledge or software proficiency (unlike flow cytometry), the ADAM(®) cytometer can be used as a rapid and reliable POCT device to measure platelet activation in peripheral blood. This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases.