Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

PubMed

Díaz-Murillo, Violeta; Medina-Estrada, Ivan; López-Meza, Joel E; Ochoa-Zarzosa, Alejandra

2016-04-01

β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian innate immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

Solar energy storage using surfactant micelles

NASA Astrophysics Data System (ADS)

Srivastava, R. C.; Marwadi, P. R.; Latha, P. K.; Bhise, S. B.

1982-09-01

The results of experiments designed to test the soluble reduced form of thionine dye as a suitable solar energy storage agent inside the hydrophobic core of surfactant micelles are discussed. Aqueous solutions of thionine, methylene blue, cetyl pyridinium bromide, sodium lauryl sulphate, iron salts, and iron were employed as samples of anionic, cationic, and nonionic surfactants. The solutions were exposed to light until the dye disappeared, and then added drop-by-drop to surfactant solutions. The resultant solutions were placed in one cell compartment while an aqueous solution with Fe(2+) and Fe(3+) ions were placed in another, with the compartments being furnished with platinum electrodes connected using a saturated KCl-agar bridge. Data was gathered on the short circuit current, maximum power, and internal resistance encountered. Results indicate that dye-surfactant systems are viable candidates for solar energy storage for later conversion to electrical power.

Effect of electrochemical redox reaction on growth and metabolism of Saccharomyces cerevisiae as an environmental factor.

PubMed

Na, Kwan Byung; Hwang, Tae Sik; Lee, Sung Hun; Ahn, Dae Hee; Park, Doo Hyun

2007-03-01

The effect of an electrochemically generated oxidation-reduction potential and electric pulse on ethanol production and growth of Saccharomyces cerevisiae ATCC 26603 was experimented and compared with effects of electron mediators (neutral red, benzyl viologen, and thionine), chemical oxidants (hydrogen peroxide and hypochlorite), chemical reductants (sulfite and nitrite), oxygen, and hydrogen. The oxidation (anodic) and reduction (cathodic) potential and electric pulse activated ethanol production and growth, and changed the total soluble protein pattern of the test strain. Neutral red electrochemically reduced activated ethanol production and growth of the test strain, but benzyl viologen and thionine did not. Nitrite inhibited ethanol production but did not influence growth of the test strain. Hydrogen peroxide, hypochlorite, and sulfite did not influence ethanol production and growth of the test strain. Hydrogen and oxygen also did not influence the growth and ethanol production. It shows that the test strain may perceive electrochemically generated oxidation-reduction potential and electric pulse as an environmental factor.

An Amperometric Immunosensor Based on Multi-Walled Carbon Nanotubes-Thionine-Chitosan Nanocomposite Film for Chlorpyrifos Detection

PubMed Central

Sun, Xia; Cao, Yaoyao; Gong, Zhili; Wang, Xiangyou; Zhang, Yan; Gao, Jinmei

2012-01-01

In this work, a novel amperometric immunosensor based on multi-walled carbon nanotubes-thionine-chitosan (MWCNTs-THI-CHIT) nanocomposite film as electrode modified material was developed for the detection of chlorpyrifos residues. The nanocomposite film was dropped onto a glassy carbon electrode (GCE), and then the anti-chlorpyrifos monoclonal antibody was covalently immobilized onto the surface of MWCNTs-THI-CHIT/GCE using the crosslinking agent glutaraldehyde (GA). The modification procedure was characterized by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimized conditions, a linear relationship between the relative change in peak current of different pulse voltammetry (DPV) and the logarithm of chlorpyrifos solution concentration was obtained in the range from 0.1 to 1.0 × 105 ng/mL with a detection limit of 0.046 ng/mL. The proposed chlorpyrifos immunosensor exhibited high reproducibility, stability, and good selectivity and regeneration, making it a potential alternative tool for ultrasensitive detection of chlorpyrifos residues in vegetables and fruits. PMID:23443396

Investigation of antimicrobial peptide genes associated with fungus and insect resistance in maize

USDA-ARS?s Scientific Manuscript database

Antimicrobial peptides (AMPs) are small defense proteins present in various organisms. Major groups of AMPs include beta-barrelin, hevein, knottin, lipid transfer protein (LTP), thionin, defensin, snakin, and cyclotide. Most plant AMPs involve host plant resistance to pathogens such as fungi, viruse...

Overexpression of a modified plant thionin enhances disease resistance to citrus canker and huanglongbing (HLB, citrus greening)

USDA-ARS?s Scientific Manuscript database

Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the United States citrus industry. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized e...

Enhancing Intrinsic Cochlear Stress Defenses to Reduce Noise-Induced Hearing Loss

DTIC Science & Technology

2002-09-01

trauma were wearing hearing protection when the hearing loss causing the accident took place. Poor fit of the protector will further degrade ...acetylcysteine and oral me- thionine in paracetamol poisoning. S Afr Med J 1986;60: 279. 133. Di Rocco A, Tagliati M, Danisi F, et al. A pilot study

Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB)

USDA-ARS?s Scientific Manuscript database

Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the United States citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an ec...

Fabrication and Optimization of Stable, Optically Transparent, and Reusable pH-Responsive Silk Membranes

PubMed Central

Toytziaridis, Andreas; Dicko, Cedric

2016-01-01

The fabrication of silk-based membranes that are stable, optically transparent and reusable is yet to be achieved. To address this bottleneck we have developed a method to produce transparent chromogenic silk patches that are optically responsive to pH. The patches were produced by blending regenerated silk fibroin (RSF), Laponite RD (nano clay) and the organic dyes neutral red and Thionine acetate. The Laponite RD played a central role in the patch mechanical integrity and prevention of dye leaching. The process was optimized using a factorial design to maximize the patch response to pH by UV absorbance and fluorescence emission. New patches of the optimized protocol, made from solutions containing 125 μM neutral red or 250 μM of Thionine and 15 mg/mL silk, were further tested for operational stability over several cycles of pH altering. Stability, performance, and reusability were achieved over the tested cycles. The approach could be extended to other reporting molecules or enzymes able to bind to Laponite. PMID:27854303

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode.

PubMed

Kong, Fen-Ying; Xu, Mao-Tian; Xu, Jing-Juan; Chen, Hong-Yuan

2011-10-15

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels. Copyright © 2011 Elsevier B.V. All rights reserved.

Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

PubMed

Wang, Xiaonan; Wang, Meiwen; Zhang, Yuanyuan; Miao, Xiaocao; Huang, Yuanyuan; Zhang, Juan; Sun, Lizhou

2016-09-15

A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability. Copyright © 2016 Elsevier B.V. All rights reserved.

EVALUATION OF IMMOBILIZED REDOX INDICATORS AS REVERSIBLE, IN SITU REDOX SENSORS FOR DETERMINING FE(III)-REDUCING CONDITIONS IN ENVIRONMENTAL SAMPLES. (R828772)

EPA Science Inventory

An in situ methodology based on immobilized redox indicators has been developed to determine when Fe(III)-reducing conditions exist in environmental systems. The redox indicators thionine (Thi, formal potential at pH 7 (E₇^0') equals 66 mV), tol...

Sensitive electrochemical sensing platform for microRNAs detection based on shortened multi-walled carbon nanotubes with high-loaded thionin.

PubMed

Deng, Keqin; Liu, Xinyan; Li, Chunxiang; Huang, Haowen

2018-05-31

The loading capacity of thionin (Thi) on shortened multi-walled carbon nanotubes (S-MWCNTs) and acidified multi-walled carbon nanotubes (A-MWCNTs) was compared. Two DNA probe fragments were designed for hybridization with microRNA-21 (miR-21), the microRNAs (miRNAs) model analyte. DNA probe 1 (P1) was assembled on Au nanoparticles (AuNPs) modified electrode. MiR-21 was captured by the pre-immobilized P1. A signal nanoprobe was synthesized by loading large amount of Thi on S-MWCNTs with covalently bonded probe 2 (P2). Owing to the large effective surface area of MWCNTs, fast electron shuttle of MWCNTs, high-loaded Thi on S-MWCNTs, and the increased conductivity from AuNPs, after signal probe hybridized with miR-21, it gave rise to a magnified current response on electrode. The increased electrochemical current enabled us to quantitatively detect miR-21. Expensive bioreagents and labeled target/detection DNA or miRNAs were avoided in this strategy. The operation complexity and assay cost were also reduced. Copyright © 2018 Elsevier B.V. All rights reserved.

Accurate counting of neurons in frozen sections: some necessary precautions.

PubMed Central

Cooper, J D; Payne, J N; Horobin, R W

1988-01-01

In 30 microns frozen sections of rat midbrain the retrograde axonal transport of diamidino yellow, a fluorescent tracer, was used to demonstrate a population of neurons in the substantia nigra. However, when visualisation was carried out using the routine Nissl method a significant proportion of neurons failed to stain. As the presence of the retrograde tracer did not affect Nissl staining of such cells, such incomplete staining, with consequent underestimation of neuronal populations, is probably a common error in similar material. Further investigation revealed that the proportion of such unstained neurons was greater when the staining time was short, when stain concentration was low, or when section thickness was increased. Some stains were worse in this respect than others. Cresyl fast violet resulted in the highest proportion of unstained neurons, thionin resulted in the lowest proportion. It was concluded that the rate of diffusion of the stain into the section was the main factor limiting the staining of neurons present. Staining with pure thionin at 0.1% concentration for at least 3 minutes and with sections no thicker than 30 microns is one regime which would avoid this problem. Images Fig. 1 PMID:2461923

H2S induced coma and cardiogenic shock in the rat: Effects of phenothiazinium chromophores

PubMed Central

SONOBE, TAKASHI; HAOUZI, PHILIPPE

2015-01-01

Context Hydrogen sulfide (H2S) intoxication produces an acute depression in cardiac contractility-induced circulatory failure, which has been shown to be one of the major contributors to the lethality of H2S intoxication or to the neurological sequelae in surviving animals. Methylene blue (MB), a phenothiazinium dye, can antagonize the effects of the inhibition of mitochondrial electron transport chain, a major effect of H2S toxicity. Objectives We investigated whether MB could affect the immediate outcome of H2S-induced coma in unanesthetized animals. Second, we sought to characterize the acute cardiovascular effects of MB and two of its demethylated metabolites—azure B and thionine—in anesthetized rats during lethal infusion of H2S. Materials and methods First, MB (4 mg/kg, intravenous [IV]) was administered in non-sedated rats during the phase of agonal breathing, following NaHS (20 mg/kg, IP)-induced coma. Second, in 4 groups of urethane-anesthetized rats, NaHS was infused at a rate lethal within 10 min (0.8 mg/min, IV). Whenever cardiac output (CO) reached 40% of its baseline volume, MB, azure B, thionine, or saline were injected, while sulfide infusion was maintained until cardiac arrest occurred. Results Seventy-five percent of the comatose rats that received saline (n = 8) died within 7 min, while all the 7 rats that were given MB survived (p = 0.007). In the anesthetized rats, arterial, left ventricular pressures and CO decreased during NaHS infusion, leading to a pulseless electrical activity within 530 s. MB produced a significant increase in CO and dP/dtmax for about 2 min. A similar effect was produced when MB was also injected in the pre-mortem phase of sulfide exposure, significantly increasing survival time. Azure B produced an even larger increase in blood pressure than MB, while thionine had no effect. Conclusion MB can counteract NaHS-induced acute cardiogenic shock; this effect is also produced by azure B, but not by thionine, suggesting that the presence of methyl groups is a prerequisite for producing this protective effect. PMID:25965774

Antifungal activity of synthetic cowpea defensin Cp-thionin II and its application in dough.

PubMed

Thery, Thibaut; Arendt, Elke K

2018-08-01

Plant defensins are small, cysteine-rich antimicrobial peptides of the immune system found in several organs during plant development. A synthetic peptide, KT43C, a linear analogue of the native Cp-thionin II found in cowpea seeds, was evaluated for its antifungal potential. It was found that KT43C displayed antifungal activity against Fusarium culmorum, Penicillium expansum and Aspergillus niger. Like native plant defensins, KT43C showed thermostability up to 100 °C and cation sensitivity. The synthetic peptide decreased the fungal growth without inducing morphogenic changes in the fungal hyphae. Non-inhibitory concentrations of the peptide induced permeabilization of the fungal membrane. In addition, high concentrations of KT43C induced the production of reactive oxygen species in the granulated cytoplasm. To investigate potential applications, the peptide was used as an additive in the preparation of dough which did not contain yeast. This peptide delayed the development of fungal growth in the dough by 2 days. Furthermore, KT43C did not induce red blood cell lysis up to a concentration of 200 μg.ml -1 . These results highlight the potential for the use of synthetic antimicrobial defensins for shelf-life extension of food products. Copyright © 2018 Elsevier Ltd. All rights reserved.

Electrografting of thionine diazonium cation onto the graphene edges and decorating with Au nano-dendrites or glucose oxidase: Characterization and electrocatalytic applications.

PubMed

Shervedani, Reza Karimi; Amini, Akbar; Sadeghi, Nima

2016-03-15

Thionine (Th) diazonium cation is covalently attached onto the glassy carbon (GC) electrode via graphene nanosheets (GNs) (GC-GNs-Th). The GC-GNs-Th electrode is subjected to further modifications to fabricate (i) glucose and (ii) nitrite sensors. Further modifications include: (i) direct immobilization of glucose oxidase (GOx) and (ii) electrodeposition of gold dendrite-like nanostructures (DGNs) on the GC-GNs-Th surface, constructing GC-GNs-Th-GOx and GC-GNs-Th-DGNs modified electrodes, respectively. The GC-GNs-Th-GOx biosensor exhibited a linear response range to glucose, from 0.5 to 6.0mM, with a limit of detection (LOD) of 9.6 μM and high sensitivity of 43.2 µAcm(-2)mM(-1). Also, the GC-GNs-Th-DGNs sensor showed a wide dynamic response range for NO2(-) ion with two linear parts, from 0.05 μM to 1.0 μM and 30.0 μM to 1.0mM, a sensitivity of 263.2 μAmM(-1) and a LOD of 0.01 μM. Applicability of the modified electrodes was successfully tested by determination of glucose in human blood serum and nitrite in water based on addition/recovery tests. Copyright © 2015 Elsevier B.V. All rights reserved.

An electrochemical immunosensor for efficient detection of uropathogenic E. coli based on thionine dye immobilized chitosan/functionalized-MWCNT modified electrode.

PubMed

Gayathri, Chandran Hema; Mayuri, Pinapeddavari; Sankaran, Krishnan; Kumar, Annamalai Senthil

2016-08-15

Uropathogenic Escherichia coli (UPEC) is the major cause of 150 million Urinary Tract Infections (UTI) reported annually world-wide. High prevalence of multi-drug-resistance makes it dangerous and difficult to cure. Therefore simple, quick and early diagnostic tools are essential for effective treatment and control. We report an electrochemical immunosensor based on thionine dye (Th) immobilized on functionalized-multiwalled carbon nanotube+chitosan composite coated on glassy carbon electrode (GCE/f-MWCNT-Chit@Th) for quick and sensitive detection of UPEC in aqueous solution. This immunosensor was constructed by sequential immobilization of UPEC, bovine serum albumin, primary antibody and Horse Radish Peroxidase (HRP) tagged secondary antibody on the surface of GCE/f-MWCNT-Chit@Th. When analyzed using 2.5mM of hydrogen peroxide reduction reaction using cyclic voltammetry in phosphate buffer, pH 7.0, the immunosensor showed excellent linearity in a range of 10(2)-10(9)cfu of UPEC mL(-1) with a current sensitivity of 7.162μA {log(cfumL(-1))}(-1). The specificity of this immunosensor was tested using other UTI and non-UTI bacteria, Staphylococcus, Klebsiella, Proteus and Shigella. The clinical applicability of the immunosensor was also successfully tested directly in UPEC spiked urine samples (simulated sample). Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles.

PubMed

Li, Guiyin; Li, Shanshan; Wang, Zhihong; Xue, Yewei; Dong, Chenyang; Zeng, Junxiang; Huang, Yong; Liang, Jintao; Zhou, Zhide

2018-04-15

Sensitive and accurate detection of tumor markers is critical to early diagnosis, point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an important clinical tumor marker for hepatocellular carcinoma (HCC), and the concentration of AFP in human serum is related to the stage of HCC. In this paper, a label-free electrochemical aptasensor for AFP detection was fabricated using AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic property and large specific surface area, RGO and Au NPs were employed on the screen-printed carbon electrode to load TH molecules. The TH not only acted as a bridging molecule to effectively capture and immobilize AFP-aptamer, but as the electron transfer mediator to provide the electrochemical signal. The AFP detection was based on the monitoring of the electrochemical current response change of TH by the differential pulse voltammetry. Under optimal conditions, the electrochemical responses were proportional to the AFP concentration in the range of 0.1-100.0 μg/mL. The limit of detection was 0.050 μg/mL at a signal-to-noise ratio of 3. The proposed method may provide a promising application of aptamer with the properties of facile procedure, low cost, high selectivity in clinic. Copyright © 2018. Published by Elsevier Inc.

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

PubMed

Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

2010-02-15

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy.

PubMed

Wang, Xiaoying; Shu, Guofang; Gao, Chanchan; Yang, Yu; Xu, Qian; Tang, Meng

2014-12-01

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs-PA6-PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs-ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Plant Antimicrobial Peptides as Potential Anticancer Agents

PubMed Central

Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo

2015-01-01

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy. PMID:25815333

Broad-spectrum protein kinase inhibition by the staurosporine analog KT-5720 reverses ethanol withdrawal-associated loss of NeuN/Fox-3.

PubMed

Reynolds, Anna R; Saunders, Meredith A; Berry, Jennifer N; Sharrett-Field, Lynda J; Winchester, Sydney; Prendergast, Mark A

2017-11-01

Chronic, intermittent ethanol (CIE) exposure is known to produce neuroadaptive alterations in excitatory neurotransmission that contribute to the development of dependence. Although activation of protein kinases (e.g., cyclic AMP [cAMP]-dependent protein kinase) is implicated in the synaptic trafficking of these receptors following CIE exposure, the functional consequences of these effects are yet to be fully understood. The present study sought to delineate the influence of protein kinase in regulating cytotoxicity following CIE exposure, as well as to examine the relative roles of ethanol exposure and ethanol withdrawal (EWD) in promoting these effects. Rat hippocampal explants were exposed to a developmental model of CIE with or without co-application of broad-spectrum protein kinase inhibitor KT-5720 (1 μM) either during ethanol exposure or EWD. Hippocampal cytotoxicity was assessed via immunofluorescence (IF) of neuron-specific nuclear protein (NeuN) with thionine staining of Nissl bodies to confirm IF findings. Concomitant application of ethanol and KT-5720 restored the loss of NeuN/Fox-3 IF in pyramidal CA1 and granule DG cell layers produced by CIE, but there was no restoration in CA3. Application of KT-5720 during EWD failed to significantly alter levels of NeuN IF, implying that ethanol exposure activates protein kinases that, in part, mediate the effects of EWD. KT-5720 application during EWD also restored thionine staining in CA1, suggesting kinase regulation of both neurons and non-neuronal cells. These data demonstrate that CIE exposure alters protein kinase activity to promote ethanol withdrawal-associated loss of NeuN/Fox-3 and highlight the influence of kinase signaling on distinct cell types in the developing hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

The monoamine oxidase inhibition properties of selected structural analogues of methylene blue.

PubMed

Delport, Anzelle; Harvey, Brian H; Petzer, Anél; Petzer, Jacobus P

2017-06-15

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC 50 =0.0037μM), Nile blue (IC 50 =0.0077μM) and 1,9-dimethyl methylene blue (IC 50 =0.018μM) exhibiting higher potency inhibition compared to MB (IC 50 =0.07μM). Nile blue also represents a potent MAO-B inhibitor with an IC 50 value of 0.012μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST. Copyright © 2017 Elsevier Inc. All rights reserved.

Electricity generation in microbial fuel cells using neutral red as an electronophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, D.H.; Zeikus, J.G.

2000-04-01

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. Inmore » microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator was 10-fold more than the amount produced when thionin was the electron mediator. The amount of electrical energy generated and the amount of current produced from glucose in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge was used in the fuel cell, stable and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Their results are discussed in relation to factors that may improve the relatively low electrical efficiencies obtained with microbial fuel cells.« less

Electron transfer reactions of excited dyes with metal complexes. Progress report, May 1, 1976--January 31, 1977. [Fe(III)--thionine reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lichtin, N.N.

1977-02-01

A study was initiated of the factors which determine quantum efficiency of transfer of reducing equivalents between excited dye molecules and metal complexes in their ground state and composition and dynamics of formation and decay of related photostationary states. A ruby laser capable of delivering a 3.6 J, 19 nsec flash was acquired and assembly of an apparatus for laser flash photolysis begun. At the same time, conventional flash photolysis was used to pursue investigation of the dependence upon solvent, anions, pH, and ionic strength of the kinetics of the spontaneous dark reaction of Fe(H/sub 2/O)/sup 3 +//sub 6/ withmore » leucothionine and with semithionine, reactions which contribute to the composition and dynamics of formation and decay of the photostationary state of the iron-thionine photoredox reaction. Results are consistent with formation of an intermediate complex between leucothionine and Fe(III), K/sub A/ = 380 M/sup -1/ and k(elec. transfer) = 0.88 s/sup -1/ at approximately 22/sup 0/ in water solution at pH2, with sulfate as anion and ..mu.. = .05 - .1 M. Under similar conditions in 50 v/v percent aqueous CH/sub 3/CN, K/sub A/ = 780 M/sup -1/, k(elec. transfer) = 0.55 s/sup -1/. In both solvents, sulfate produces a large positive salt effect. Intermediacy of a complex was not established for the faster reaction of Fe(III) with semithionine under similar conditions: K/sub A/ . k(elec. transfer) approximately 3.5 x 10/sup 5/ M/sup -1/s/sup -1/ in H/sub 2/O, approximately 1.0 x 10/sup 4/ in 50 v/v percent aqueous CH/sub 3/CN.« less

Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.

PubMed

Taveira, Gabriel B; Mello, Érica O; Carvalho, André O; Regente, Mariana; Pinedo, Marcela; de La Canal, Laura; Rodrigues, Rosana; Gomes, Valdirene M

2017-05-01

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL -1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H 2 O 2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms. © 2017 Wiley Periodicals, Inc.

The photokilling of bladder carcinoma cells in vitro by phenothiazine dyes.

PubMed

Fowler, G J; Rees, R C; Devonshire, R

1990-09-01

The potential photodynamic therapy photosensitizers Methylene Blue, Azure C, Methylene Violet, Thionine, Methylene Green, Haematoporphyrin, Nile Blue A, chloroaluminium phthalocyanine and bis-aluminium phthalocyanine were examined for their photoeffects and dark toxicity against a human superficial bladder carcinoma cell-line. By examination of [3H]thymidine uptake into dye-treated cells after irradiation with a copper-vapour pumped dye laser, it was found that Methylene Blue was the most phototoxic and dark toxic of all the dyes tested, suggesting that the dye might be of some use as a topically applied photodrug for use in photodynamic therapy of superficial or early-recurring carcinomas.

Nitrogen fertilization and plant growth promoting rhizobacteria treatments affected amino acid content of cabbage

NASA Astrophysics Data System (ADS)

Dursun, Atilla; Yildirim, Ertan; Ekinci, Melek; Turan, Metin; Kul, Raziye; Karagöz, Fazilet P.

2017-04-01

This study was designed to determine the influence of a nitrogen fixing plant growth promoting rhizobacteria (PGPR) inoculation (seed coating and seedling dipping) and 6 doses of nitrogen (0, 40, 80, 120, 160, 200 kg ha-1) application on amino acid contents of cabbage. Coating and seedling dipping applications caused a significant increase in values histidine, glycine, thionin, arginine and alanine of cabbage. Highest glutamate, serine, asparagines and glutamine contents were obtained from 160-200 kg ha-1 nitrogen dose applied plants. As a result, the use of bacteria treatments provides means of improving amino acid contents in cabbage.

News from the Biological Stain Commission.

PubMed

Lyon, H O; Kiernan, J A

2008-12-01

In the three earlier editions of News from the Biological Stain Commission (BSC), under the heading of "Regulatory affairs," the BSC's International Affairs Committee reported on the work of Technical Committee 212, Clinical Laboratory Testing and in Vitro Diagnostic Test Systems of the International Standards Organization (ISO/TC 212) and its working groups, WG 1, WG 2 and WG 3. In this issue of News from the BSC, H.O. Lyon provides information from the annual meeting of ISO/TC 212 that took place June 2-4, 2008 in Vancouver, British Columbia, Canada. In addition, under the heading of "Certification," J.A. Kiernan examines the certification procedure for thionine used by the BSC laboratory in Rochester, NY.

Electricity Generation in Microbial Fuel Cells Using Neutral Red as an Electronophore

PubMed Central

Park, Doo Hyun; Zeikus, J. Gregory

2000-01-01

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells. PMID:10742202

A dual enzymatic-biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages of diabetic mice: evaluation of the diabetes-accelerated atherosclerosis risk.

PubMed

Huang, Qilin; An, Yarui; Tang, Linlin; Jiang, Xiaoli; Chen, Hua; Bi, Wenji; Wang, Zhongchuan; Zhang, Wen

2011-11-30

In this paper, a novel dual enzymatic-biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), and ChOx, were covalently attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The developed biosensor had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from 0.008 mM to 6.0 mM for glucose with a detection limit of 2.0 μM, and a linear range from 0.002 mM to 1.0 mM for cholesterol with a detection limit of 0.6 μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis. Copyright © 2011 Elsevier B.V. All rights reserved.

The monoamine oxidase inhibition properties of selected structural analogues of methylene blue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delport, Anzelle

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, themore » present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC{sub 50} = 0.0037 μM), Nile blue (IC{sub 50} = 0.0077 μM) and 1,9-dimethyl methylene blue (IC{sub 50} = 0.018 μM) exhibiting higher potency inhibition compared to MB (IC{sub 50} = 0.07 μM). Nile blue also represents a potent MAO-B inhibitor with an IC{sub 50} value of 0.012 μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST. - Highlights: • MB analogues, cresyl violet and Nile blue, are high potency MAO-A inhibitors. • Nile blue also represents a potent MAO-B inhibitor. • Potent MAO-A inhibition should alert to potential serotonin toxicity.« less

Sensitization of photoprocesses in colloidal Ag2S quantum dots by dye molecules

NASA Astrophysics Data System (ADS)

Ovchinnikov, Oleg V.; Kondratenko, Tamara S.; Grevtseva, Irina G.; Smirnov, Mikhail S.; Pokutnyi, Sergey I.

2016-07-01

The effect of photosensitization of IR luminescence excitation (1205 nm) of colloidal Ag2S quantum dots (QDs) with average size of 2.5±0.6 nm in gelatin at 600 to 660 nm by molecules of 3,3'-di-(γ-sulfopropyl)-4,4',5,5'-dibenzo-9-ethylthiacarbocyanine betaine pyridinium salt (Dye1) and thionine dye (Dye2) was registered. Cis-J-aggregates of Dye1 and cations monomer of Dye2 conjugated with Ag2S QDs take part in this process. The photosensitization of luminescence excitation of colloidal Ag2S QDs was interpreted by resonance nonradiation transfer of electronic excitation energy from cis-J-aggregates of Dye1 and cations of Dye2 to centers of recombination luminescence of Ag2S QDs.

Photogalvanic cells driven by electron transfer quenching of excited singlet states

NASA Astrophysics Data System (ADS)

Creed, D.; Fawcett, N. C.

Photoreduction of oxonine by iron(II) sulfate in dilute acid is produced by quenching of the excited signlet state (S1). No induced intersystem crossing to the tripolet (T1) is observed by nanosecond flash photolysis. The photoreduction of oxonine (S1) by iron(II) was used in a totally illuminated thin layer photogalvanic cell. Power conversion efficiencies are, however, very low. The fluorescence of oxonine and thiazine dyes such as thionine is quenched by acids. Oxonine fluorescence is also quenched by hydroquinone and catechol sulfonates and related compounds. Eleven new thiazine dyes were synthesized. A few photogalvanic experiments were carried out using high concentrations of the water miscible dye and iron(II) in a TI/TL cell. Ferrophos, an iron phosphorus alloy, can be substituted for platinum or gold as a cathode in photogalvanic cells.

Fenton-treated functionalized diamond nanoparticles as gene delivery system.

PubMed

Martín, Roberto; Alvaro, Mercedes; Herance, José Raúl; García, Hermenegildo

2010-01-26

When raw diamond nanoparticles (Dnp, 7 nm average particle size) obtained from detonation are submitted to harsh Fenton-treatment, the resulting material becomes free of amorphous soot matter and the process maintains the crystallinity, reduces the particle size (4 nm average particle size), increases the surface OH population, and increases water solubility. All these changes are beneficial for subsequent Dnp covalent functionalization and for the ability of Dnp to cross cell membranes. Fenton-treated Dnps have been functionalized with thionine and the resulting sample has been observed in HeLa cell nuclei. A triethylammonium-functionalized Dnp pairs electrostatically with a plasmid having the green fluorescent protein gene and acts as gene delivery system permitting the plasmid to cross HeLa cell membrane, something that does not occur for the plasmid alone without assistance of polycationic Dnp.

Changes in functional metabolism in the rat central nervous system following spaceflight

NASA Technical Reports Server (NTRS)

Murakami, D. M.; Miller, J. D.; Fuller, C. A.

1985-01-01

The neuronal metabolism and soma size of neurons within the paraventricular nucleus (PVN) and the supraoptic nucleus of rats are analyzed. Five male Sprague-Dawley rats were flown on Spacelab 3 for 7 days under a 12:12 light/dark cycle and unlimited food and water, and a control group was kept on the ground under similar conditions. The preparation of the hypothalamus of the rats for microscopic examination using thionin or the cytochrome oxidase (CYOX) technique is described. CYOX activity and soma size within the PVN are evaluated. The effects of water drinking pattern and space flight on CYOX activity and soma size are investigated. The data reveal that the flight rats with normal drinking patterns display a decrease in neuronal metabolism within the vasopressin-containing neurons of the hypothalamus and this metabolic change may reflect fluid shifts caused by microgravity.

A sex difference in the hypothalamic uncinate nucleus: relationship to gender identity.

PubMed

Garcia-Falgueras, Alicia; Swaab, Dick F

2008-12-01

Transsexuality is an individual's unshakable conviction of belonging to the opposite sex, resulting in a request for sex-reassignment surgery. We have shown previously that the bed nucleus of the stria terminalis (BSTc) is female in size and neuron number in male-to-female transsexual people. In the present study we investigated the hypothalamic uncinate nucleus, which is composed of two subnuclei, namely interstitial nucleus of the anterior hypothalamus (INAH) 3 and 4. Post-mortem brain material was used from 42 subjects: 14 control males, 11 control females, 11 male-to-female transsexual people, 1 female-to-male transsexual subject and 5 non-transsexual subjects who were castrated because of prostate cancer. To identify and delineate the nuclei and determine their volume and shape we used three different stainings throughout the nuclei in every 15th section, i.e. thionin, neuropeptide Y and synaptophysin, using an image analysis system. The most pronounced differences were found in the INAH3 subnucleus. Its volume in thionin sections was 1.9 times larger in control males than in females (P < 0.013) and contained 2.3 times as many cells (P < 0.002). We showed for the first time that INAH3 volume and number of neurons of male-to-female transsexual people is similar to that of control females. The female-to-male transsexual subject had an INAH3 volume and number of neurons within the male control range, even though the treatment with testosterone had been stopped three years before death. The castrated men had an INAH3 volume and neuron number that was intermediate between males (volume and number of neurons P > 0.117) and females (volume P > 0.245 and number of neurons P > 0.341). There was no difference in INAH3 between pre-and post-menopausal women, either in the volume (P > 0.84) or in the number of neurons (P < 0.439), indicating that the feminization of the INAH3 of male-to-female transsexuals was not due to estrogen treatment. We propose that the sex reversal of the INAH3 in transsexual people is at least partly a marker of an early atypical sexual differentiation of the brain and that the changes in INAH3 and the BSTc may belong to a complex network that may structurally and functionally be related to gender identity.

Antimicrobial activity of gamma-thionin-like soybean SE60 in E. coli and tobacco plants.

PubMed

Choi, Yeonhee; Choi, Yang Do; Lee, Jong Seob

2008-10-17

The SE60, a low molecular weight, sulfur-rich protein in soybean, is known to be homologous to wheat gamma-purothionin. To elucidate the functional role of SE60, we expressed SE60 cDNA in Escherichia coli and in tobacco plants. A single protein band was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after anti-FLAG affinity purification of the protein from transformed E. coli. While the control E. coli cells harboring pFLAG-1 showed standard growth with Isopropyl beta-d-1-thiogalactopyranoside (IPTG) induction, E. coli cells expressing the SE60 fusion protein did not grow at all, suggesting that SE60 has toxic effects on E. coli growth. Genomic integration and the expression of transgene in the transgenic tobacco plants were confirmed by Southern and Northern blot analysis, respectively. The transgenic plants demonstrated enhanced resistance against the pathogen Pseudomonas syringae. Taken together, these results strongly suggest that SE60 has antimicrobial activity and play a role in the defense mechanism in soybean plants.

Comparative anatomy of the accessory ciliary ganglion in mammals.

PubMed

Kuchiiwa, S; Kuchiiwa, T; Suzuki, T

1989-01-01

The orbits of 13 mammalian species (pig, sika deer, domestic sheep, horse, cat, fox, racoon dog, marten, rat, rabbit, crab-eating macaque, japanese macaque and man) were stained with silver nitrate and dissected under a dissecting microscope with special attention to the presence and location of the accessory ciliary ganglion. Some preparations were stained with thionin and examined as whole-mounts in a transmission microscope. The accessory ciliary ganglion was present in all 13 species, although the number and degree of development varied greatly from species to species. The accessory ciliary ganglion could be readily differentiated from the main ciliary ganglion in the following respects: it was located on the short ciliary nerve, and it had no root derived directly from the inferior trunk of the oculomotor nerve and it never attaches to this nerve. In many species, ganglion cells were also scattered in the short ciliary nerves in the stained whole preparations. In a few species, there were one or more small ganglia on the nerve to the inferior oblique muscle.

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production.

PubMed

Dowd, Patrick F; Johnson, Eric T

2015-05-01

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease resistance-associated genes in milk stage kernels from commercial popcorn fields over 3 years. Relatively lower expression of resistance gene types was noted in years with higher temperatures and lower rainfall, which was consistent with prior results for many previously identified resistance response-associated genes. The lower rates of expression occurred for genes such as chitinases, protease inhibitors, and peroxidases; enzymes involved in the synthesis of cell wall barriers and secondary metabolites; and regulatory proteins. However, expression of several specific resistance genes previously associated with mycotoxins, such as aflatoxin in dent maize, was not affected. Insect damage altered the spectrum of resistance gene expression differences compared to undamaged ears. Correlation analyses showed expression differences of some previously reported resistance genes that were highly associated with mycotoxin levels and included glucanases, protease inhibitors, peroxidases, and thionins.

Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.).

PubMed

Fujimura, Masatoshi; Ideguchi, Mineo; Minami, Yuji; Watanabe, Keiichi; Tadera, Kenjiro

2004-03-01

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

Increased electrocatalyzed performance through high content potassium doped graphene matrix and aptamer tri infinite amplification labels strategy: Highly sensitive for matrix metalloproteinases-2 detection.

PubMed

Ren, Xiang; Zhang, Tong; Wu, Dan; Yan, Tao; Pang, Xuehui; Du, Bin; Lou, Wanruo; Wei, Qin

2017-08-15

Herein, a super-labeled immunoassay was fabricated for matrix metalloproteinases-2 detection. A self-corrosion ITO micro circuit board was designed in this sensing platform to reduce the random error in the same testing condition, and the self-constructed sensing platform is portable with a cheap price. The K-modified graphene (K-GS) was utilized as the matrix material, which was synthesized well by phenylate and phenanthrene through the polar bond of nonpolar molecule phenylate and the π-π interaction for the first time. An aptamer-based labels based on Au nanoparticles (AuNPs), thionine (Th) and horseradish peroxidase (HRP) were applied as the signal source for tri infinite amplification. This fabricated super-labeled immunoassay exhibit excellent performance for MMPs-2 detection. It displayed a broad linear range of 10 -4 -10ng/mL with a low detection limit of 35 fg/mL, which may have a potential application in the clinical diagnose. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis.

PubMed

López-Solanilla, E; García-Olmedo, F; Rodríguez-Palenzuela, P

1998-06-01

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat alpha-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.

Ce(III, IV)-MOF electrocatalyst as signal-amplifying tag for sensitive electrochemical aptasensing.

PubMed

Yu, Hua; Han, Jing; An, Shangjie; Xie, Gang; Chen, Sanping

2018-06-30

Metal-organic frameworks (MOFs) as a new class of porous materials have attracted increasing attention in the field of biomimetic catalysis. This study firstly reports a mixed valence state Ce-MOF possessing intrinsic catalytic activity towards thionine (Thi), and its application in constructing an amplified electrochemical aptasensor for thrombin detection. As noticed, the novel catalytic process combines the advantages of 3D infinite extension of the Ce(III, IV)-MOF skeleton containing large amounts of catalytic sites and spontaneous recycling of the Ce(III)/Ce(IV) for electrochemical reduction of Thi, thereby presenting amplified electrochemical signals. To further improve the aptasensor performance, the high selectivity of proximity binding-induced DNA strand displacement and high efficiency of exonuclease III-assisted recycling amplification were incorporated into the assay. The aptasensor was employed to detect thrombin in complex serum samples, which shows high sensitivity, specificity, stability and reproducibility. This work offers an opportunity to develop MOF-based electrocatalyst as signal-amplifying tag for versatile bioassays and catalytic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

PubMed

Kraszewska, Joanna; Beckett, Michael C; James, Tharappel C; Bond, Ursula

2016-07-15

Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A ratiometric strategy -based electrochemical sensing interface for the sensitive and reliable detection of imidacloprid.

PubMed

Li, Xueyan; Kan, Xianwen

2018-04-30

In this study, a ratiometric strategy-based electrochemical sensor was developed by electropolymerization of thionine (THI) and β-cyclodextrin (β-CD) composite films on a glassy carbon electrode surface for imidacloprid (IMI) detection. THI played the role of an inner reference element to provide a built-in correction. In addition, the modified β-CD showed good selective enrichment for IMI to improve the sensitivity and anti-interference ability of the sensor. The current ratio between IMI and THI was calculated as the detected signal for IMI sensing. Compared with common single-signal sensing, the proposed ratiometric strategy showed a higher linear range and a lower limit of detection of 4.0 × 10-8-1.0 × 10-5 mol L-1 and 1.7 × 10-8 mol L-1, respectively, for IMI detection. On the other hand, the ratiometric strategy endowed the sensor with good accuracy, reproducibility, and stability. The sensor was also used for IMI determination in real samples with satisfactory results. The simple, effective, and reliable way reported in this study can be further used to prepare ratiometric strategy-based electrochemical sensors for the selective and sensitive detection of other compounds with good accuracy and stability.

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds.

PubMed

Deharo, E; García, R N; Oporto, P; Gimenez, A; Sauvain, M; Jullian, V; Ginsburg, H

2002-04-01

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.

THE CYTOLOGY AND GENETICS OF RADIATION RESISTANCE IN BACTERIA. Progress Report for the Period of November 1, 1955 to November 1, 1956

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

1960-10-31

Cytogenetic procedures, applicable to microbiology, were selected and tested on a suitable organism as a basis for the valid application of these procedures to other microorganisms. Nocardia corallina was chosen as a test organism on the basis of preliminary cytological studies. The crystal violet nuclear stain, the thionin-SO/sub 2/ nuclear stain, the crystal violet-tannic acid-congo red cell wall stain, and phase microscopy, were found to be valid tools of microbial cytology if interpreted with restraint. The correlation of cytological and radiobiological findings demonstrated that, in N. corallina, a diploid coccoidal stage, gives rise to a coenocytic diploid hypbal stage whichmore » fragments through a nuclear reduction division to form haploid dinucleated bacillary cells. The bacillary cell nuclei fuse and the cell divides to form diploid coccoids. The haploid chromosome number is suggested as three for this organism. It has been demonstrated that a microbial cytogenetic approach involving the correlation and integration of cytological procedures with genetic and radiobiological methods can aid in solving basic problems of microbial cytology and genetics. (For preceding period see ORO-282.) (auth)« less

Pseudothionin-St1, a potato peptide active against potato pathogens.

PubMed

Moreno, M; Segura, A; García-Olmedo, F

1994-07-01

A 5-kDa polypeptide, pseudothionin Solanum tuberosum 1 (Pth-St1), which was active against Clavibacter michiganensis subspecies sepedonicus, a bacterial pathogen of potatoes, has been purified from the buffer-insoluble fraction of potato tubers by salt extraction and HPCL. Pth-St1 was also active against other potato pathogens tested (Pseudomonas solanacearum and Fusarium solani). The N-terminal amino acid sequence of this peptide was identical (except for a N/H substitution at position 2) to that deduced from a previously reported cDNA sequence (EMBL accession number X-13180), which had been misclassified as a Browman-Birk protease inhibitor. Pth-St1 did not inhibit either trypsin or insect alpha-amylase activities, and, in contrast with true thionins, did not affect cell-free protein synthesis or beta-glucuronidase activity. Northern-blot and tissue-print analyses showed that steady-state mRNA levels were highest in flowers (especially in petals), followed by tubers (especially in the epidermal cell layers and in leaf primordia), stems and leaves. Infection of leaves with a bacterial pathogen suspended in 10 mM MgCl2 switched off the gene, whereas mock inoculation with 10 mM MgCl2 alone induced higher mRNA levels.

"Sign-on/off" sensing interface design and fabrication for propyl gallate recognition and sensitive detection.

PubMed

Dai, Yunlong; Li, Xueyan; Fan, Limei; Lu, Xiaojing; Kan, Xianwen

2016-12-15

A new strategy based on sign-on and sign-off was proposed for propyl gallate (PG) determination by an electrochemical sensor. The successively modified poly(thionine) (PTH) and molecular imprinted polymer (MIP) showed an obvious electrocatalysis and a good recognition toward PG, respectively. Furthermore, the rebound PG molecules in imprinted cavities not only were oxidized but also blocked the electron transmission channels for PTH redox. Thus, a sign-on from PG current and a sign-off from PTH current were combined as a dual-sign for PG detection. Meanwhile, the modified MIP endowed the sensor with recognition capacity. The electrochemical experimental results demonstrated that the prepared sensor possessed good selectivity and high sensitivity. A linear ranging from 5.0×10(-8) to 1.0×10(-4)mol/L for PG detection was obtained with a limit of detection of 2.4×10(-8)mol/L. And the sensor has been applied to analyze PG in real samples with satisfactory results. The simple, low cost, and effective strategy reported here can be further used to prepare electrochemical sensors for other compounds selective recognition and sensitive detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Salivary gland oncocytes in African hedgehogs (Atelerix albiventris) mimicking cytomegalic inclusion disease.

PubMed

Brunnert, S R; Hensley, G T; Citino, S B; Herron, A J; Altman, N H

1991-07-01

The salivary glands from three African hedgehogs contained multiple foci of cytomegalic cells, which occasionally had a mild to moderate infiltrate of lymphocytes at the periphery. The cytomegalic cells were 35 to 40 microns in diameter with abundant acidophilic granular to hyalin cytoplasm. The nuclei were enlarged with clumped marginalized chromatin and a large, (6 to 8 microns in diameter) central, brightly eosinophilic nucleolus that had the appearance of an inclusion body by light microscopy. Histochemically most of the cytomegalic cells contained cytoplasmic metachromatic granules with Feyrter's thionine inclusion stain. Scattered cells at the periphery of the cytomegalic foci contained periodic acid-Schiff-positive cytoplasmic granules. Ultrastructurally the cytomegalic cells contained numerous tightly-packed, often bizarre, enlarged mitochondria that completely filled the cytoplasm. The nucleus consisted of a dense central core of chromatin associated with the nucleolus and the remaining chromatin was clumped and marginalized. Nuclear and cytoplasmic virions consistent with cytomegalovirus were not present. Histochemical stains of the nucleus for heavy metals were negative. The ultrastructural and histochemical findings of the cytomegalic cells were consistent with oncocytes. Previous reports in the literature of similar cells in the salivary glands of insectivores appear to have been erroneously described as cytomegalovirus infections.

High-performance electrochemical mercury aptasensor based on synergistic amplification of Pt nanotube arrays and Fe3O4/rGO nanoprobes.

PubMed

Luo, Jingyi; Jiang, Danfeng; Liu, Tao; Peng, Jingmeng; Chu, Zhenyu; Jin, Wanqin

2018-05-01

In this work, a novel sandwich-type aptasensor was designed for the ultrasensitive recognition of trace mercury ions in water. Numerous oriented platinum nanotube arrays (PtNAs) were in-situ crystallized on a flexible electrode as a sensing interface, while thionine labelled Fe 3 O 4 /rGO nanocomposites as signal amplifiers. Both PtNAs/CF and nanocomposites were synthesized by easy hydrothermal processes. With their large surface area, it was favorable for electrochemical performance and immobilization of capture DNAs (cDNA) and report DNAs (rDNA). Upon the existence of Hg 2+ , partial linker DNAs were tightly bound with cDNAs through thymine-Hg 2+ -thymine pairing (T-Hg 2+ -T). Then rDNAs attached Fe 3 O 4 /rGO nanoprobes were fixed on the electrode through the match of remaining linker DNAs and rDNAs. Under the optimal conditions, the Hg 2+ aptasensor showed a synergistic amplification performance with a wide linear range from 0.1nM to 100nM, as well as a low detection limit of 30pM. Moreover, the as-prepared aptasensor also exhibited reliable performance for assay in real lake water samples. Copyright © 2017. Published by Elsevier B.V.

Mucinous (colloid) adenocarcinomas secrete distinct O-acylated forms of sialomucins: a histochemical study of gastric, colorectal and breast adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Poveda, M A; Segura, D I

2001-12-01

Mucinous (colloid) adenocarcinomas represent a distinct group of tumours defined by the presence of large amounts of extracellular mucins. By using histochemical methods, we analysed mucins secreted by mucinous versus non-mucinous adenocarcinomas and looked for differential secretion profiles. Sixty-four adenocarcinomas were studied (23 colorectal, 17 gastric, and 24 breast tumours). Thirty-two tumours were of the colloid type. The following methods were applied to paraffin tissue sections: (i) Alcian blue (pH 2.5) and periodic acid-Schiff (PAS); (ii) high iron diamine and Alcian blue (pH 2.5); (iii) periodic acid borohydride, potassium hydroxide, and PAS; (iv) periodic acid-thionine Schiff, potassium hydroxide, and PAS; and (v) periodic acid-borohydride and PAS. Most adenocarcinomas secreted acidic mucins, with sialomucins predominating over sulfomucins, except for non-mucinous adenocarcinomas of the breast which showed predominant neutral mucins. All mucinous adenocarcinomas contained C9-O-acyl sialic acid as mono, di(C8,C9)-, or tri(C7,C8,C9)-O-acyl forms. Acidic mucins secreted by the majority of non-colloid adenocarcinomas consisted of non-O-acylated sialomucins. C9-O-acylation of sialic acid is a characteristic feature of mucinous adenocarcinomas and can be readily detected by histochemical methods.

Antimicrobial Peptides from Plants

PubMed Central

Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

2015-01-01

Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

Morphological and micromorphological characteristics of Desmodium fruits (Leguminosae: Papilionoideae).

PubMed

Freitas, Daiane M; Reis, Ademir; Bortoluzzi, Roseli L da Costa; Santos, Marisa

2014-12-01

The genus Desmodium is represented in Santa Catarina State, Brazil, by 13 species, all with lomen- taceous fruits. Shape, size and isthmus margin of loments vary, while the surface is glabrous, or covered by trichomes of different types. Morphological diversity of trichomes becomes particularly relevant to taxonomic description. The trichome types present on the surface of Desmodium fruits provide data for the identification and classification of species in the State. To assess this, three fruits of each species were collected and deposited at two herbaria, HBR and FLOR, in Santa Catarina, Brazil. Some rehydrated samples were examined using light microscopy (LM); and some sections were exposed to the following histochemical reagents: Sudan III for oils and Thionine for mucilage. The structural aspects of trichomes can be classified into uni- or multicellular and may still be simple, i.e., nonglandular or glandular. Using scanning electron microscopy (SEM), five types of trichomes were identified and analyzed among the Desmodium species studied: uncinate, uniseriate, globose multicellular, globose unicellular and subulate. Characteristics, such as loment margin and article form, glabrescent or pillous indument, trichome type, with or without papillous epidermal cells and epicuticular striations, showed relevant diagnostic value. An identification key was developed for Desmodium species from Santa Catarina State, Brazil, based on macro and micromorphological characters of the fruit.

Application of fractal and grey level co-occurrence matrix analysis in evaluation of brain corpus callosum and cingulum architecture.

PubMed

Pantic, Igor; Dacic, Sanja; Brkic, Predrag; Lavrnja, Irena; Pantic, Senka; Jovanovic, Tomislav; Pekovic, Sanja

2014-10-01

This aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.

Electrochemical immunoassay for carcinoembryonic antigen based on signal amplification strategy of nanotubular mesoporous PdCu alloy.

PubMed

Cai, Yanyan; Li, He; Li, Yuyang; Zhao, Yanfang; Ma, Hongmin; Zhu, Baocun; Xu, Caixia; Wei, Qin; Wu, Dan; Du, Bin

2012-01-01

Interests in using nanoporous metals for biosensing applications have been increasing. Herein, nanotubular mesoporous PdCu (NM-PdCu) alloy is used to fabricate a novel label-free electrochemical immunosensor for cancer biomarker carcinoembryonic antigen (CEA). It operates through physisorption of anti-CEA on NM-PdCu and the mixture of sulfonated graphene sheets (HSO(3)-GS) and thionine (TH) functionalized glassy carbon electrode interface as the detection platform. In this study, chitosan (CS)-PdCu is bound very strongly to carcinoembryonic antibody (anti-CEA), because of the good electron conductivity, high surface area, and good biocompatibility. CS-PdCu is immobilized on electrodes by electrostatic interactions between the negatively charged sulfo group of HSO(3)-GS and the abundant positively charged amino groups of chitosan. TH acts as the redox probe. Under the optimized conditions, the electrochemical immunosensor exhibits a wide working range from 0.01 to 12 ng/mL with a low detection limit of 4.86 pg/mL. The accuracy, reproducibility, and stability of the immunosensor are acceptable. The assay is evaluated for real serum samples, receiving satisfactory results. The nanoporous metal materials-based immunoassay provides a promising approach in clinical application and thus represents a versatile detection method. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of intermittent hypobaric hypoxia preconditioning on the expression of neuroglobin and Bcl-2 in the rat hippocampal CA1 area following ischemia-reperfusion.

PubMed

Wu, Q; Yu, K X; Ma, Q S; Liu, Y N

2015-09-09

This study was aimed at understanding the effect of intermittent hypobaric hypoxia preconditioning (IHHP) on neuroglobin (NGB) and Bcl-2 expression in the hippocampal CA1 region of rats following global cerebral ischemia-reperfusion. Wistar rats were randomly divided into sham, IHHP control, global cerebral ischemia-reperfusion (IR group), and IHHP+IR groups. The four-vessel occlusion rat model of Pulsinelli was used for the IR groups, in which the common carotid artery was occluded for 8 min before reperfusion. Thionin and immunohistochemical staining were used to observe NGB and Bcl-2 expression in the hippocampal CA1 region. Data was analyzed using the SPSS software. There was a significant increase in the number of surviving cells in the hippocampal CA1 region of the IHHP+IR group (119.5 ± 14) compared to the IR group (41.7 ± 3.8) (P < 0.05). There was a significant increase in the expression of NGB and Bcl-2 in the hippocampal CA1 region of the IHHP+IR group compared to the IR group. By upregulating hippocampal NGB and Bcl-2 expression, IHHP may play a role in neural protection by reducing hippocampal neuronal apoptosis following IR.

Use of one- and two-mediator systems for developing a BOD biosensor based on the yeast Debaryomyces hansenii.

PubMed

Zaitseva, A S; Arlyapov, V A; Yudina, N Yu; Alferov, S V; Reshetilov, A N

2017-03-01

We investigated the use of one- and two-mediator systems in amperometric BOD biosensors (BOD, biochemical oxygen demand) based on the yeast Debaryomyces hansenii. Screening of nine mediators potentially capable of electron transfer - ferrocene, 1,1'-dimethylferrocene, ferrocenecarboxaldehyde, ferroceneacetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methylene blue and potassium ferricyanide - showed only ferrocene and neutral red to be efficient electron carriers for the eukaryotes studied. Two-mediator systems based on combinations of the investigated compounds were used to increase the efficiency of electron transfer. The developed two-mediator biosensors exceeded their one-mediator analogs by their characteristics. The most preferable two-mediator system for developing a BOD biosensor was a ferrocene-methylene blue combination that ensured a satisfactory long-time stability (43 days), selectivity, sensitivity (the lower limit of the determined BOD 5 concentrations, 2.5mg О 2 /dm 3 ) and speed (assay time for one sample, not greater than 10min) of BOD determination. Analysis of water samples showed that the use of a ferrocene-methylene blue two-mediator system and the yeast D. hansenii enabled registration of data that highly correlated with the results of the standard method (R=0.9913). Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

PubMed

Tang, Dianping; Tang, Juan; Su, Biling; Chen, Guonan

2010-10-27

A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

PubMed

Agarwal, Nitin; Biancardi, Alberto M; Patten, Florence W; Reeves, Anthony P; Seibel, Eric J

2014-04-01

Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

Cytological study of DNA content and nuclear morphometric analysis for aid in the diagnosis of high-grade dysplasia within oral leukoplakia.

PubMed

Yang, Xi; Xiao, Xuan; Wu, Wenyan; Shen, Xuemin; Zhou, Zengtong; Liu, Wei; Shi, Linjun

2017-09-01

To quantitatively examine the DNA content and nuclear morphometric status of oral leukoplakia (OL) and investigate its association with the degree of dysplasia in a cytologic study. Oral cytobrush biopsy was carried out to obtain exfoliative epithelial cells from lesions before scalpel biopsy at the same location in a blinded series of 70 patients with OL. Analysis of nuclear morphometry and DNA content status using image cytometry was performed with oral smears stained with the Feulgen-thionin method. Nuclear morphometric analysis revealed significant differences in DNA content amount, DNA index, nuclear area, nuclear radius, nuclear intensity, sphericity, entropy, and fractal dimension (all P < .01) between low-grade and high-grade dysplasia. DNA content analysis identified 34 patients with OL (48.6%) with DNA content abnormality. Nonhomogeneous lesion (P = .018) and high-grade dysplasia (P = .008) were significantly associated with abnormal DNA content. Importantly, the positive correlation between the degree of oral dysplasia and DNA content status was significant (P = .004, correlation coefficient = 0.342). Cytology analysis of DNA content and nuclear morphometric status using image cytometry may support their use as a screening and monitoring tool for OL progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

PubMed Central

Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

"Reticular" and "Areticular" Nissl Bodies in Sympathetic Neurons of a Lizard

PubMed Central

Smith, Stuart W.

1959-01-01

Sympathetic ganglia of the horned lizard, Phrynosoma cornutum, were fixed in OsO4 and imbedded in methacrylate. Thin sections were cut for electron microscopy. Some adjacent thick sections were cut for light microscopy and were stained in acidified, dilute thionine both before and after digestion by RNase. In the light microscope two types of Nissl bodies are found, both removable by RNase: (1) a deep, diffuse, indistinctly bounded, metachromatic variety, and (2) a superficial, dense, sharply delimited, orthochromatic sort. Electron microscopically, the former ("reticular" Nissl bodies) corresponds to the granulated endoplasmic reticular structure of Nissl material previously described by others, whereas the latter ("areticular" Nissl bodies) comprises compact masses of particles of varying internal density and devoid of elements of endoplasmic reticulum. The constituent particles of the areticular Nissl material are 4 to 8 x the diameter of single ribonucleoprotein granules of the reticular Nissl substance and seem, near zones of junction with the reticular type, to arise by clustering of such granules with subsequent partial dispersion of the substance of the granules into an added, less dense material. It is suggested that the observed orthochromasia of the areticular Nissl substance is due to accumulation of a large amount of protein bound to RNA and, further, that these Nissl bodies may represent storage depots of RNA and protein. PMID:13673051

A sandwich-type electrochemical immunoassay for ultrasensitive detection of non-small cell lung cancer biomarker CYFRA21-1.

PubMed

Zeng, Yan; Bao, Jing; Zhao, Yanan; Huo, Danqun; Chen, Mei; Qi, Yanli; Yang, Mei; Fa, Huanbao; Hou, Changjun

2018-04-01

Many studies confirm that the aberrant expression of Cytokeratin 19 fragment 21-1 (CYFRA21-1) is highly correlated with non-small cell lung cancer (NSCLC), especially for squamous cell carcinoma. Herein, we report a sandwich-type electrochemical immunosensor based on signal amplification strategy of multiple nanocomposites to test CYFRA21-1 selectively and sensitively. The proposed immunosensor fabricated by three-dimensional graphene (3D-G), chitosan (CS) and glutaraldehyde (GA) composite on the glass carbon electrode (GCE) with a large surface area is prepared to immobilize primary antibodies (Ab 1 ) and provide excellent conductivity. To further amplify the electrochemical signal, the trace tag on the foundation of gold nanoparticles (AuNPs) is coated with amino-functionalized carbon nanotube (MWCNT-NH 2 ) nanocomposite through thionine linking, which provides more amino groups to capture more horseradish peroxidase-labeled antibodies (HPR-Ab 2 ) and enhances the conductivity. Under optimal conditions, the developed immunosensor exhibits excellent analytical performance for the determination of CYFRA21-1 with a wide linear range from 0.1 to 150ng·mL -1 and a low detection limit (LOD) of 43pg·mL -1 . Furthermore, satisfactory results are obtained for the determination of CYFRA21-1 in real clinical serum samples, indicating the potential of the immunoassay to be applied in clinical analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

PubMed

Martínez, Diana; Thompson, Carolina; Draghi, Graciela; Canavesio, Vilma; Jacobo, Roberto; Zimmer, Patricia; Elena, Sebastián; Nicola, Ana M; de Echaide, Susana Torioni

2014-09-17

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas. Copyright © 2014 Elsevier B.V. All rights reserved.

Activation of p38 MAPK participates in brain ischemic tolerance induced by limb ischemic preconditioning by up-regulating HSP 70.

PubMed

Sun, Xiao-Cai; Xian, Xiao-Hui; Li, Wen-Bin; Li, Li; Yan, Cai-Zhen; Li, Qing-Jun; Zhang, Min

2010-08-01

This study investigates whether activation of p38 MAPK by the up-regulation of HSP 70 participates in the induction of brain ischemic tolerance by limb ischemic preconditioning (LIP). Western blot and immunohistochemical assays indicated that p38 MAPK activation occurred earlier than HSP 70 induction in the CA1 region of the hippocampus after LIP. P-p38 MAPK expression was up-regulated at 6h and reached its peak 12h after LIP, while HSP 70 expression was not significantly increased until 1 day and peaked 2 days after LIP. Neuropathological evaluation by thionin staining showed that quercetin (4 ml/kg, 50mg/kg, intraperitoneal injection), an inhibitor of HSP 70, blocked the protective effect of LIP against delayed neuronal death that is normally induced by lethal brain ischemic insult, indicating that HSP 70 participates in the induction of brain ischemic tolerance by LIP. Furthermore, SB 203580, an inhibitor of HSP 70, inhibited HSP 70 activation in the CA1 region of the hippocampus induced by LIP either with or without the presence of subsequent brain ischemic insult. Based on the above results, it can be concluded that activation of p38 MAPK participates in the brain ischemic tolerance induced by LIP at least partly by the up-regulation of HSP 70 expression. (c) 2010 Elsevier Inc. All rights reserved.

Sensitive electrochemical immunosensor for α-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels.

PubMed

An, Yarui; Jiang, Xiaoli; Bi, Wenji; Chen, Hua; Jin, Litong; Zhang, Shengping; Wang, Chuangui; Zhang, Wen

2012-02-15

A novel electrochemical immunosensor for sensitive detection of α-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab(2)-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab(2)) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the {HRP-Ab(2)-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H(2)O(2)) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and {HRP-Ab(2)-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes. Copyright © 2011 Elsevier B.V. All rights reserved.

Glucose Biosensor Based on a Glassy Carbon Electrode Modified with Polythionine and Multiwalled Carbon Nanotubes

PubMed Central

Tang, Wenwei; Li, Lei; Wu, Lujun; Gong, Jiemin; Zeng, Xinping

2014-01-01

A novel glucose biosensor was fabricated. The first layer of the biosensor was polythionine, which was formed by the electrochemical polymerisation of the thionine monomer on a glassy carbon electrode. The remaining layers were coated with chitosan-MWCNTs, GOx, and the chitosan-PTFE film in sequence. The MWCNTs embedded in FAD were like “conductive wires” connecting FAD with electrode, reduced the distance between them and were propitious to fast direct electron transfer. Combining with good electrical conductivity of PTH and MWCNTs, the current response was enlarged. The sensor was a parallel multi-component reaction system (PMRS) and excellent electrocatalytic performance for glucose could be obtained without a mediator. The glucose sensor had a working voltage of −0.42 V, an optimum working temperature of 25°C, an optimum working pH of 7.0, and the best percentage of polytetrafluoroethylene emulsion (PTFE) in the outer composite film was 2%. Under the optimised conditions, the biosensor displayed a high sensitivity of 2.80 µA mM−1 cm−2 and a low detection limit of 5 µM (S/N = 3), with a response time of less than 15 s and a linear range of 0.04 mM to 2.5 mM. Furthermore, the fabricated biosensor had a good selectivity, reproducibility, and long-term stability, indicating that the novel CTS+PTFE/GOx/MWCNTs/PTH composite is a promising material for immobilization of biomolecules and fabrication of third generation biosensors. PMID:24816121

Glucose biosensor based on a glassy carbon electrode modified with polythionine and multiwalled carbon nanotubes.

PubMed

Tang, Wenwei; Li, Lei; Wu, Lujun; Gong, Jiemin; Zeng, Xinping

2014-01-01

A novel glucose biosensor was fabricated. The first layer of the biosensor was polythionine, which was formed by the electrochemical polymerisation of the thionine monomer on a glassy carbon electrode. The remaining layers were coated with chitosan-MWCNTs, GOx, and the chitosan-PTFE film in sequence. The MWCNTs embedded in FAD were like "conductive wires" connecting FAD with electrode, reduced the distance between them and were propitious to fast direct electron transfer. Combining with good electrical conductivity of PTH and MWCNTs, the current response was enlarged. The sensor was a parallel multi-component reaction system (PMRS) and excellent electrocatalytic performance for glucose could be obtained without a mediator. The glucose sensor had a working voltage of -0.42 V, an optimum working temperature of 25°C, an optimum working pH of 7.0, and the best percentage of polytetrafluoroethylene emulsion (PTFE) in the outer composite film was 2%. Under the optimised conditions, the biosensor displayed a high sensitivity of 2.80 µA mM(-1) cm(-2) and a low detection limit of 5 µM (S/N = 3), with a response time of less than 15 s and a linear range of 0.04 mM to 2.5 mM. Furthermore, the fabricated biosensor had a good selectivity, reproducibility, and long-term stability, indicating that the novel CTS+PTFE/GOx/MWCNTs/PTH composite is a promising material for immobilization of biomolecules and fabrication of third generation biosensors.

Localisation of the spinal nucleus of the accessory nerve in the rabbit.

PubMed Central

Ullah, M; Salman, S S

1986-01-01

The spinal nucleus of the accessory nerve (SNA) was localised in eight adult rabbits by a retrograde degeneration technique using thionine as a stain for the Nissl substance. The SNA was found to extend from the caudal one fifth of the medulla oblongata to the cranial one fourth of the sixth cervical segment. In the caudal part of the medulla oblongata, the SNA was located in the dorsal part of the detached ventral grey column. In the first cervical segment, the SNA was dorsolateral to the dorsomedial column and dorsal to the ventromedial column of the ventral grey column. In the cranial part of the second cervical segment, the SNA shifted laterally to the lateral margin of the ventral grey column. After this lateral shift, the SNA was located in the lateral part of the ventral grey column of the second, third and fourth cervical segments. In the fifth and cranial one fourth of the sixth cervical segments, the SNA was not a well defined column of cells but was represented by isolated cells scattered in the ventral part of the ventral grey column between the phrenic nucleus and the ventral border of the grey matter. The total number of chromatolysed cells found in the SNA of the right experimental side varied from 2723 to 3210. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 PMID:3429311

Magnetic dispersive solid-phase extraction based on graphene oxide/Fe3 O4 @polythionine nanocomposite followed by atomic absorption spectrometry for zinc monitoring in water, flour, celery and egg.

PubMed

Babaei, Azar; Zeeb, Mohsen; Es-Haghi, Ali

2018-07-01

Magnetic graphene oxide nanocomposite has been proposed as a promising and sustainable sorbent for the extraction and separation of target analytes from food matrices. Sample preparation based on nanocomposite presents several advantages, such as desired efficiency, reasonable selectivity and high surface-area-to-volume ratio. A new graphene oxide/Fe 3 O 4 @polythionine (GO/Fe 3 O 4 @PTh) nanocomposite sorbent was introduced for magnetic dispersive solid-phase extraction and flame atomic absorption spectrometric detection of zinc(II) in water, flour, celery and egg. To fabricate the sorbent, an oxidative polymerization of thionine on the surface of magnetic GO was applied, while polythionine was simply employed as a surface modifier to improve extraction yield. The properties of the sorbent were characterized by transmission electron microscopy, scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray analysis, vibrating sample magnetometry and Fourier transform-infrared spectroscopy. The calibration curve showed linearity in the range of 0.5-30 ng mL -1 . Limits of detection (S/N = 3) and quantification (S/N = 10) were 0.08 and 0.5 ng mL -1 , respectively. The method was applied for trace-level determination of Zn(II) in water and food samples, and its validation was investigated by recovery experiments and analyzing certified reference material. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

An organic-inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels.

PubMed

Su, Biling; Tang, Dianping; Tang, Juan; Li, Qunfang; Chen, Guonan

2011-10-01

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic-inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core-shell Fe(3)O(4)-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H(2)O(2) as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001-200 ng mL(-1) AFP with a low detection limit of 0.5 pg mL(-1) at 3s(B). Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL(-1) AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay. Copyright © 2011 Elsevier Inc. All rights reserved.

A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein.

PubMed

Lu, Dingqiang; Xu, Qiuda; Pang, Guangchang; Lu, Fuping

2018-06-05

An electrochemical double-layer Au nanoparticle membrane immunosensor was developed using an electrochemical biosensing signal amplification system with Au nanoparticles, thionine, chitosan, and horseradish peroxidase, which was fabricated using double self-adsorption of Au nanoparticle sol followed by anti-α-fetoprotein Balb/c mouse monoclonal antibody adsorption. The AuNPs sol was characterized by spectrum scanning and transmission electron microscopy. The immunosensor was characterized by atomic force microscopy, cyclic voltammetry, and alternating-current impedance during each stage of adsorption and assembly. The amperometric I-t curve method was used to measure α-fetoprotein (AFP) diluted in phosphate buffered saline. The result indicated a wide linear range, and the change rate of steady-current before and after immune response had linear correlation within the range 0.1-10 4  pg/mL AFP. The current change rate equation was △I = 5.82334 lgC + 37.01195 (R 2  = 0.9922). The lowest limit of detection was 0.03 pg/mL (S/N = 3), and the reproducibility of the sensor was good. Additionally, the sensor could be stably stored above phosphate buffered saline at 4 °C for more than 24 days. More importantly, the sensor is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling.

Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues.

PubMed

Li, Ye Long; Dai, Xin Ren; Yue, Xun; Gao, Xin-Qi; Zhang, Xian Sheng

2014-10-01

Maize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. Partial SSP gene expressions in reproductive tissues were determined by qRT-PCR. Small secreted peptides (SSPs) are important cell-cell communication messengers in plants. Most information on plant SSPs come from Arabidopsis thaliana and Oryza sativa, while little is known about the SSPs of other grass species such as maize (Zea mays). In this study, we identified 1,491 SSP genes from maize genomic sequences. These putative SSP genes were distributed throughout the ten maize chromosomes. Among them, 611 SSPs were classified into 198 superfamilies according to their conserved domains, and 725 SSPs with four or more cysteines at their C-termini shared similar cysteine arrangements with their counterparts in other plant species. Moreover, the SSPs requiring post-translational modification, as well as defensin-like (DEFL) proteins, were identified. Further, the expression levels of 110 SSP genes were analyzed in reproductive tissues, including male flower, pollen, silk, and ovary. Most of the genes encoding basal-layer antifungal peptide-like, small coat proteins-like, thioredoxin-like proteins, γ-thionins-like, and DEFL proteins showed high expression levels in the ovary and male flower compared with their levels in silk and mature pollen. The rapid alkalinization factor-like genes were highly expressed only in the mature ovary and mature pollen, and pollen Ole e 1-like genes showed low expression in silk. The results of this study provide basic information for further analysis of SSP functions in the reproductive process of maize.

Graphene-palladium nanowires based electrochemical sensor using ZnFe2O4-graphene quantum dots as an effective peroxidase mimic.

PubMed

Liu, Weiyan; Yang, Hongmei; Ma, Chao; Ding, Ya-nan; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2014-12-10

We proposed an electrochemical DNA sensor by using peroxidase-like magnetic ZnFe2O4-graphene quantum dots (ZnFe2O4/GQDs) nanohybrid as a mimic enzymatic label. Aminated graphene and Pd nanowires were successively modified on glassy carbon electrode, which improved the electronic transfer rate as well as increased the amount of immobilized capture ssDNA (S1). The nanohybrid ZnFe2O4/GQDs was prepared by assembling the GQDs on the surface of ZnFe2O4 through a photo-Fenton reaction, which was not only used as a mimic enzyme but also as a carrier to label complementary ssDNA (S3). By synergistically integrating highly catalytically activity of nano-sized GQDs and ZnFe2O4, the nanohybrid possessed highly-efficient peroxidase-like catalytic activity which could produce a large current toward the reduction of H2O2 for signal amplification. Thionine was used as an excellent electron mediator. Compared with traditional enzyme labels, the mimic enzyme ZnFe2O4/GQDs exhibited many advantages such as environment friendly and better stability. Under the optimal conditions, the approach provided a wide linear range from 10(-16) to 5×10(-9) M and low detection limit of 6.2×10(-17) M. The remarkable high catalytic capability could allow the nanohybrid to replace conventional peroxidase-based assay systems. The new, robust and convenient assay systems can be widely utilized for the identification of other target molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

PubMed

Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

2006-10-01

Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.

Effect of 2,450 MHz microwave radiation on the development of the rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inouye, M.; Galvin, M.J.; McRee, D.I.

1983-12-01

Male Sprague-Dawley rats were exposed to 2,450 MHz microwave radiation at an incident power density of 10 mW/cm2 daily for 3 hours from day 4 of pregnancy (in utero exposure) through day 40 postpartum, except for 2 days at the perinatal period. The animals were killed, and the brains removed, weighed, measured, and histologically examined at 15, 20, 30, and 40 days of age. The histologic parameters examined included the cortical architecture of the cerebral cortex, the decline of the germinal layer along the lateral ventricles, the myelination of the corpus callosum, and the decline of the external germinal layermore » of the cerebellar cortex. In 40-day-old rats, quantitative measurements of neurons were also made. The spine density of the pyramidal cells in layer III of the somatosensory cortex, and the density of basal dendritic trees of the pyramidal cells in layer V were measured in Golgi-Cox impregnated specimens. In addition, the density of Purkinje cells and the extent of the Purkinje cell layer in each lobule were measured in midsagittal sections of the cerebellum stained with thionin. There were no remarkable differences between microwave-exposed and control (sham-irradiated) groups for any of the histologic or quantitative parameters examined; however, the findings provide important information on quantitative measurements of the brain. The data from this study failed to demonstrate that there is a significant effect on rat brain development due to microwave exposure (10 mW/cm2) during the embryonic, fetal, and postnatal periods.« less

Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance.

PubMed

Ali, Sajad; Ganai, Bashir Ahmad; Kamili, Azra N; Bhat, Ajaz Ali; Mir, Zahoor Ahmad; Bhat, Javaid Akhter; Tyagi, Anshika; Islam, Sheikh Tajamul; Mushtaq, Muntazir; Yadav, Prashant; Rawat, Sandhya; Grover, Anita

Pathogenesis-related (PR) proteins and antimicrobial peptides (AMPs) are a group of diverse molecules that are induced by phytopathogens as well as defense related signaling molecules. They are the key components of plant innate immune system especially systemic acquired resistance (SAR), and are widely used as diagnostic molecular markers of defense signaling pathways. Although, PR proteins and peptides have been isolated much before but their biological function remains largely enigmatic despite the availability of new scientific tools. The earlier studies have demonstrated that PR genes provide enhanced resistance against both biotic and abiotic stresses, which make them one of the most promising candidates for developing multiple stress tolerant crop varieties. In this regard, plant genetic engineering technology is widely accepted as one of the most fascinating approach to develop the disease resistant transgenic crops using different antimicrobial genes like PR genes. Overexpression of PR genes (chitinase, glucanase, thaumatin, defensin and thionin) individually or in combination have greatly uplifted the level of defense response in plants against a wide range of pathogens. However, the detailed knowledge of signaling pathways that regulates the expression of these versatile proteins is critical for improving crop plants to multiple stresses, which is the future theme of plant stress biology. Hence, this review provides an overall overview on the PR proteins like their classification, role in multiple stresses (biotic and abiotic) as well as in various plant defense signaling cascades. We also highlight the success and snags of transgenic plants expressing PR proteins and peptides. Copyright © 2018 Elsevier GmbH. All rights reserved.

Proteomic analysis of S-nitrosylated and S-glutathionylated proteins in wheat seedlings with different dehydration tolerances.

PubMed

Gietler, Marta; Nykiel, Małgorzata; Orzechowski, Sławomir; Fettke, Joerg; Zagdańska, Barbara

2016-11-01

A loss of dehydration tolerance in wheat seedlings on the fifth day following imbibition is associated with a disturbance in cellular redox homeostasis, as documented by a shift of the reduced/oxidized glutathione ratio to a more oxidized state and a significant increase in the ratio of protein thiols to the total thiol group content. Therefore, the identification and characterization of redox-sensitive proteins are important steps toward understanding the molecular mechanisms of the loss of dehydration tolerance. In the present study, proteins that were differentially expressed between fully turgid (control), dehydrated tolerant (four-day-old) and dehydrated sensitive (six-day-old) wheat seedlings were analysed. Protein spots having at least a significant (p < 0.05) two-fold change in protein abundance were selected by Delta2D as differentially expressed, identified by MALDI-TOF and LC-MS/MS, and classified according to their function. The observed changes in the proteomic patterns of the differentially S-nitrosylated and S-glutathionylated proteins were highly specific in dehydration-tolerant and -sensitive wheat seedlings. The metabolic function of these proteins indicates that dehydration tolerance is mainly related to nucleic acids, protein metabolism, and energy metabolism. It has been proven that leaf-specific thionins BTH6 and DB4, chloroplastic 50S ribosomal protein L16, phospholipase A1-II delta, and chloroplastic thioredoxin M2 are both S-nitrosylated and S-glutathionylated upon water deficiency. Our results revealed the existence of interplay between S-nitrosylation and S-glutathionylation, two redox-regulated protein posttranslational modifications that could enhance plant defence mechanisms and/or facilitate the acclimation of plants to unfavourable environmental conditions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Location of the motoneurons of the mylohyoid muscle in the rat. A fluorescence and Nissl study.

PubMed

Badran, Darwish H; Al-Hadidi, Maher T; Ramadan, Hassan N; Abu-Ghaida, Jamal H

2005-01-01

To locate the neuronal motor cells of the mylohyoid muscle and discuss their topographical organization. The present study was conducted at the Department of Anatomy and Histology, Faculty of Medicine, University of Jordan, Amman, Jordan between 2002 and 2003. The mylohyoid muscle in 15 albino rats was injected with 15 mliter of a retrogradely transported fluorescent material DAPI-Pr. After a survival period of 48 hours, animals were sacrificed, fixed in situ and brains harvested. The caudorostral transverse sections of the hindbrains were examined under the fluorescence microscope to detect the fluorescing cells, which were immediately photographed. Sections containing the labeled cells were charted, stained with 1% thionine and photographs obtained through light and fluorescence microscopes at different magnifications. The place and shape of all labeled cells were singled out by asset of their charted referring photographs of hindbrain sections, which display the entire motor trigeminal nucleus. The results showed that the fluorescent cell increase was found to occupy the rostromedial part of the ipsilateral motor trigeminal nucleus. The nucleus was large at its caudal third; the labeled cells are mainly those of the medial "subgroup". These cells are rationally distinct and lie alongside the internal loop of the facial nerve. At the middle third, most of the medial "subgroup" was found labeled. At its middle, the nucleus found was well developed, attained an appreciable size and its medial "subgroup" was somewhat distinct. Whereas, at the rostral third, the nucleus was larger, the medial group was more distinct and all cells were labeled. The medial cellular mass of the nucleus showed reduced labeled cells at the rostral end. This study demonstrates that the rostromedial part of the motor trigeminal nucleus represents the absolute territorial domain of the mylohyoid muscle motoneurons.

Effects of treadmill exercise on behavioral recovery and neural changes in the substantia nigra and striatum of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse

PubMed Central

Goldberg, Natalie R.S.; Meshul, Charles K.

2011-01-01

Our goal was to extend our understanding of the neural changes behind motor recovery with treadmill exercise in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse. We determined the extent of dopamine (DA) terminal changes using western immunoblotting [striatal dopamine transporter (DAT) and tyrosine hydroxylase (TH)] and alterations in the mean number of DA cells/section by immunohistochemistry and Nissl staining [TH-labeled cells and thionin-stained cells in the substantia nigra pars compacta (SN-PC)]. We measured recovery of gait performance and amount of spontaneous physical activity using the parallel rod activity chamber (PRAC). We hypothesized that the decrease in TH-labeled neurons in the SN-PC due to MPTP will be partially reversed by treadmill exercise, leading to recovery of motor behavior as measured by the PRAC. Following MPTP or vehicle administration, mice ran on the treadmill for 1 hour per day at 18 cm/s, 5 days per week. Results showed that treadmill exercise improves gait performance and increases physical activity while promoting increased protein expression of striatal DAT and TH. Exercise was effective for all mice, however effects of early treadmill-based intervention appear to have an additional and unique benefit in mice who received MPTP. We are the first to show that, even following a nearly 50% decrease in the mean number of TH-labeled neurons/section in the SN-PC following MPTP, treadmill exercise leads to an increase of neurons in the SN-PC and improved motor behavior. PMID:21315689

Synthesis and characterization of thermally stable and/or conductive polymers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gajiwala, H.M.

1992-01-01

Eight new thermally stable polyimides were synthesized from two tricyclic heterocyclic diamines: thionine which has a phenothiazine moiety and proflavine which has an acridine unit. The polymerization reactions were optimized with respect to solvents, reaction time, reaction temperature, solid contents, etc., and their structure property relationships were studied. All these soluble polyimides have nice film forming properties. One of the polyimides containing the acridine moiety, appears to have a tendency to form a liquid crystalline state when its solution is passed through a fine capillary. All of these polyimides were thermally stable in air up to 500-550[degrees]C and up tomore » 600[degrees]C in a nitrogen atmosphere. They have refractive indices in the range of 1.65 to 1.85 and have relatively low value of permittivity. Two other thermally stable polymers, viz., polybenzimidazole and the ladder polymer having the phenazine moiety in the backbone were also synthesized. For these polymerization reactions, tetraamino derivative of phenazine was synthesized from the commercially available diamino, dinitro derivative of benzene. The polybenzimidazole was prepared via the azomethine pathway. This polymer had an intrinsic viscosity of 0.94 in methanesulfonic acid. The nice film forming polybenzimidazole polymer was found to be thermally stable up to 400[degrees]C. The ladder type of a polymer was synthesized by condensation polymerization between tetraaminophenazine and dihydroxybenzophenone in polyphosphoric acid at an elevated temperature. The completely conjugated ladder polymer was found to be semiconducting on doping with iodine. This polymer was highly crystalline as demonstrated by its X-ray diffraction pattern. A morphology study of the polymer indicated that the material has a tendency to form dendritic crystals. The polymer was thermally stable up to about 400[degrees]C in air.« less

Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

PubMed Central

Barua, Anita; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Prakash, Archana; Tiwari, Sapana; Arora, Sonia; Sathyaseelan, Kannusamy

2016-01-01

Background & objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis. Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization. Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M. Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis. PMID:27488010

Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model.

PubMed

Bos, P K; van Osch, G J; Frenz, D A; Verhaar, J A; Verwoerd-Verhoef, H L

2001-05-01

The ability of cartilage to regenerate following injury is limited, potentially leading to osteoarthritis. Integrative cartilage repair, necessary for durable restoration of cartilage lesions, can be regarded as a wound healing process. Little is known about the effects of growth factors regulating acute cartilage wound healing in vivo. In this study the temporal expression patterns of growth factors and proteoglycan content in cartilage wound edges in vivo were studied. Cartilage wounds were created in rabbit ear cartilage using a 6 mm biopsy punch. Specimens were subsequently harvested 1, 3, 7, 14 and 28 days after surgery. Paraffin sections were thionin stained to visualize proteoglycan loss and replacement. Immunohistochemical staining of TGFbeta1, TGFbeta3, IGF-1, IGF-II and FGF-2 was used to define growth factor expression at the cartilage wound sites. Almost no effect of cartilage wounding was observed one day after surgery. A decrease of proteoglycan content, with a maximal loss at day 7, and a subsequent restoration was observed at the wound edges. Growth factor expression increased simultaneously. Maximal immunostaining for IGF1, IGFII, FGF2 and TGF-beta3 was observed at day 7, followed by a gradual decrease. Increased expression of TGFbeta1 lasted from day 3 until day 14. We have demonstrated the ability of chondrocytes to increase growth factor expression and to restore the rapid decrease in proteoglycan content in the initial phase following acute wounding. A temporal increase in intracellular growth factor expression suggests an autocrine and/or paracrine metabolic stimulation, which can be regarded a sign of chondrocytes repair capacity. Copyright 2001 OsteoArthritis Research Society International.

Identification of ischemic regions in a rat model of stroke.

PubMed

Popp, Anke; Jaenisch, Nadine; Witte, Otto W; Frahm, Christiane

2009-01-01

Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia. Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed. TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study.

Identification of Ischemic Regions in a Rat Model of Stroke

PubMed Central

Popp, Anke; Jaenisch, Nadine; Witte, Otto W.; Frahm, Christiane

2009-01-01

Background Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia. Methodology/Principal Findings Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed. Conclusions/Significance TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study. PMID:19274095

Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

1998-12-01

Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated sialomucins in C9. Adenocarcinomas of the colloid type, thought to bear a poor prognosis, are the most heavily O-acylated.

Transcriptomic and histological responses of African rice (Oryza glaberrima) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots

PubMed Central

Petitot, Anne-Sophie; Kyndt, Tina; Haidar, Rana; Dereeper, Alexis; Collin, Myriam; de Almeida Engler, Janice; Gheysen, Godelieve

2017-01-01

Abstract Background and Aims The root-knot nematode Meloidogyne graminicola is responsible for production losses in rice (Oryza sativa) in Asia and Latin America. The accession TOG5681 of African rice, O. glaberrima, presents improved resistance to several biotic and abiotic factors, including nematodes. The aim of this study was to assess the cytological and molecular mechanisms underlying nematode resistance in this accession. Methods Penetration and development in M. graminicola in TOG5681 and the susceptible O. sativa genotype ‘Nipponbare’ were compared by microscopic observation of infected roots and histological analysis of galls. In parallel, host molecular responses to M. graminicola were assessed by root transcriptome profiling at 2, 4 and 8 d post-infection (dpi). Specific treatments with hormone inhibitors were conducted in TOG5681 to assess the impact of the jasmonic acid and salicylic acid pathways on nematode penetration and reproduction. Key Results Penetration and development of M. graminicola juveniles were reduced in the resistant TOG5681 in comparison with the susceptible accession, with degeneration of giant cells observed in the resistant genotype from 15 dpi onwards. Transcriptome changes were observed as early as 2 dpi, with genes predicted to be involved in defence responses, phenylpropanoid and hormone pathways strongly induced in TOG5681, in contrast to ‘Nipponbare’. No specific hormonal pathway could be identified as the major determinant of resistance in the rice-nematode incompatible interaction. Candidate genes proposed as involved in resistance to M. graminicola in TOG5681 were identified based on their expression pattern and quantitative trait locus (QTL) position, including chalcone synthase, isoflavone reductase, phenylalanine ammonia lyase, WRKY62 transcription factor, thionin, stripe rust resistance protein, thaumatins and ATPase3. Conclusions This study provides a novel set of candidate genes for O. glaberrima resistance to nematodes and highlights the rice-M. graminicola pathosystem as a model to study plant-nematode incompatible interactions. PMID:28334204

Potential electron mediators to extract electron energies of RBC glycolysis for prolonged in vivo functional lifetime of hemoglobin vesicles.

PubMed

Kettisen, Karin; Bülow, Leif; Sakai, Hiromi

2015-04-15

Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.

Similar, but different: structurally related azelaic acid and hexanoic acid trigger differential metabolomic and transcriptomic responses in tobacco cells.

PubMed

Djami-Tchatchou, Arnaud T; Ncube, Efficient N; Steenkamp, Paul A; Dubery, Ian A

2017-11-29

Plants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense. Metabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza. The results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates.

First isolation and characterization of Brucella microti from wild boar.

PubMed

Rónai, Zsuzsanna; Kreizinger, Zsuzsa; Dán, Ádám; Drees, Kevin; Foster, Jeffrey T; Bányai, Krisztián; Marton, Szilvia; Szeredi, Levente; Jánosi, Szilárd; Gyuranecz, Miklós

2015-07-11

Brucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary. The B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 μg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile. Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity in this Brucella species.

The Lateral Organ Boundaries Domain Transcription Factor LBD20 Functions in Fusarium Wilt Susceptibility and Jasmonate Signaling in Arabidopsis1[W

PubMed Central

Thatcher, Louise F.; Powell, Jonathan J.; Aitken, Elizabeth A.B.; Kazan, Kemal; Manners, John M.

2012-01-01

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) gene family encodes plant-specific transcriptional regulators functioning in organ development. In a screen of Arabidopsis (Arabidopsis thaliana) sequence-indexed transferred DNA insertion mutants, we found disruption of the LOB DOMAIN-CONTAINING PROTEIN20 (LBD20) gene led to increased resistance to the root-infecting vascular wilt pathogen Fusarium oxysporum. In wild-type plants, LBD20 transcripts were barely detectable in leaves but abundant in roots, where they were further induced after F. oxysporum inoculation or methyl jasmonate treatment. Induction of LBD20 expression in roots was abolished in coronatine insensitive1 (coi1) and myc2 (allelic to jasmonate insensitive1) mutants, suggesting LBD20 may function in jasmonate (JA) signaling. Consistent with this, expression of the JA-regulated THIONIN2.1 (Thi2.1) and VEGETATIVE STORAGE PROTEIN2 (VSP2) genes were up-regulated in shoots of lbd20 following treatment of roots with F. oxysporum or methyl jasmonate. However, PLANT DEFENSIN1.2 expression was unaltered, indicating a repressor role for LBD20 in a branch of the JA-signaling pathway. Plants overexpressing LBD20 (LBD20-OX) had reduced Thi2.1 and VSP2 expression. There was a significant correlation between increased LBD20 expression in the LBD20-OX lines with both Thi2.1 and VSP2 repression, and reduced survival following F. oxysporum infection. Chlorosis resulting from application of F. oxysporum culture filtrate was also reduced in lbd20 leaves relative to the wild type. Taken together, LBD20 is a F. oxysporum susceptibility gene that appears to regulate components of JA signaling downstream of COI1 and MYC2 that are required for full elicitation of F. oxysporum- and JA-dependent responses. To our knowledge, this is the first demonstration of a role for a LBD gene family member in either biotic stress or JA signaling. PMID:22786889

Transcriptomic and histological responses of African rice (Oryza glaberrima) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots.

PubMed

Petitot, Anne-Sophie; Kyndt, Tina; Haidar, Rana; Dereeper, Alexis; Collin, Myriam; de Almeida Engler, Janice; Gheysen, Godelieve; Fernandez, Diana

2017-03-01

The root-knot nematode Meloidogyne graminicola is responsible for production losses in rice ( Oryza sativa ) in Asia and Latin America. The accession TOG5681 of African rice, O. glaberrima , presents improved resistance to several biotic and abiotic factors, including nematodes. The aim of this study was to assess the cytological and molecular mechanisms underlying nematode resistance in this accession. Penetration and development in M. graminicola in TOG5681 and the susceptible O. sativa genotype 'Nipponbare' were compared by microscopic observation of infected roots and histological analysis of galls. In parallel, host molecular responses to M. graminicola were assessed by root transcriptome profiling at 2, 4 and 8 d post-infection (dpi). Specific treatments with hormone inhibitors were conducted in TOG5681 to assess the impact of the jasmonic acid and salicylic acid pathways on nematode penetration and reproduction. Penetration and development of M. graminicola juveniles were reduced in the resistant TOG5681 in comparison with the susceptible accession, with degeneration of giant cells observed in the resistant genotype from 15 dpi onwards. Transcriptome changes were observed as early as 2 dpi, with genes predicted to be involved in defence responses, phenylpropanoid and hormone pathways strongly induced in TOG5681, in contrast to 'Nipponbare'. No specific hormonal pathway could be identified as the major determinant of resistance in the rice-nematode incompatible interaction. Candidate genes proposed as involved in resistance to M. graminicola in TOG5681 were identified based on their expression pattern and quantitative trait locus (QTL) position, including chalcone synthase, isoflavone reductase, phenylalanine ammonia lyase, WRKY62 transcription factor, thionin, stripe rust resistance protein, thaumatins and ATPase3. This study provides a novel set of candidate genes for O. glaberrima resistance to nematodes and highlights the rice- M. graminicola pathosystem as a model to study plant-nematode incompatible interactions. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Electrochemical K-562 cells sensor based on origami paper device for point-of-care testing.

PubMed

Ge, Shenguang; Zhang, Lina; Zhang, Yan; Liu, Haiyun; Huang, Jiadong; Yan, Mei; Yu, Jinghua

2015-12-01

A low-cost, simple, portable and sensitive paper-based electrochemical sensor was established for the detection of K-562 cell in point-of-care testing. The hybrid material of 3D Au nanoparticles/graphene (3D Au NPs/GN) with high specific surface area and ionic liquid (IL) with widened electrochemical windows improved the good biocompatibility and high conductivity was modified on paper working electrode (PWE) by the classic assembly method and then employed as the sensing surface. IL could not only enhance the electron transfer ability but also provide sensing recognition interface for the conjugation of Con A with cells, with the cell capture efficiency and the sensitivity of biosensor strengthened simultaneously. Concanavalin A (Con A) immobilization matrix was used to capture cells. As proof-of-concept, the paper-based electrochemical sensor for the detection of K-562 cells was developed. With such sandwich-type assay format, K-562 cells as model cells were captured on the surface of Con A/IL/3D AuNPs@GN/PWE. Con A-labeled dendritic PdAg NPs were captured on the surface of K-562 cells. Such dendritic PdAg NPs worked as catalysts promoting the oxidation of thionine (TH) by H2O2 which was released from K-562 cells via the stimulation of phorbol 12-myristate-13-acetate (PMA). Therefore, the current signal response was dependent on the amount of PdAg NPs and the concentration of H2O2, the latter of which corresponded with the releasing amount from cells. So, the detection method of K-562 cell was also developed. Under optimized experimental conditions, 1.5×10(-14) mol of H2O2 releasing from each cell was calculated. The linear range and the detection limit for K-562 cells were determined to be 1.0×10(3)-5.0×10(6) cells/mL and 200 cells/mL, respectively. Such as-prepared sensor showed excellent analytical performance with good fabrication reproducibility, acceptable precision and satisfied accuracy, providing a novel protocol in point-of-care testing of cells. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA Ploidy Measured on Archived Pretreatment Biopsy Material May Correlate With Prostate-Specific Antigen Recurrence After Prostate Brachytherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keyes, Mira, E-mail: mkeyes@bccancer.bc.ca; MacAulay, Calum; Hayes, Malcolm

Purpose: To explore whether DNA ploidy of prostate cancer cells determined from archived transrectal ultrasound-guided biopsy specimens correlates with disease-free survival. Methods and Materials: Forty-seven failures and 47 controls were selected from 1006 consecutive low- and intermediate-risk patients treated with prostate {sup 125}I brachytherapy (July 1998-October 2003). Median follow-up was 7.5 years. Ten-year actuarial disease-free survival was 94.1%. Controls were matched using age, initial prostate-specific antigen level, clinical stage, Gleason score, use of hormone therapy, and follow-up (all P nonsignificant). Seventy-eight specimens were successfully processed; 27 control and 20 failure specimens contained more than 100 tumor cells were used formore » the final analysis. The Feulgen-Thionin stained cytology samples from archived paraffin blocks were used to determine the DNA ploidy of each tumor by measuring integrated optical densities. Results: The samples were divided into diploid and aneuploid tumors. Aneuploid tumors were found in 16 of 20 of the failures (80%) and 8 of 27 controls (30%). Diploid DNA patients had a significantly lower rate of disease recurrence (P=.0086) (hazard ratio [HR] 0.256). On multivariable analysis, patients with aneuploid tumors had a higher prostate-specific antigen failure rate (HR 5.13). Additionally, those with “excellent” dosimetry (V100 >90%; D90 >144 Gy) had a significantly lower recurrence rate (HR 0.25). All patients with aneuploid tumors and dosimetry classified as “nonexcellent” (V100 <90%; D90 <144 Gy) (5 of 5) had disease recurrence, compared with 40% of patients with aneuploid tumors and “excellent” dosimetry (8 of 15). In contrast, dosimetry did not affect the outcome for diploid patients. Conclusions: Using core biopsy material from archived paraffin blocks, DNA ploidy correctly classified the majority of failures and nonfailures in this study. The results suggest that DNA ploidy can be used as a useful marker for aggressiveness of localized prostate cancer. A larger study will be necessary to further confirm our hypothesis.« less

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

PubMed Central

Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S.; Brew, Simon D.; Perrett, Lorraine L.; Koylass, Mark S.; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C.; Dick, Edward J.; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E.

2014-01-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60T and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60T and F8/08-61 could be distinguished clearly from all known species of the genus Brucellaand their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucellasuggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60T ( = NCTC 13660T = CIRMBP 0958T). PMID:25242540

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

PubMed

Whatmore, Adrian M; Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S; Brew, Simon D; Perrett, Lorraine L; Koylass, Mark S; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C; Dick, Edward J; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E

2014-12-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)). Crown Copyright 2014. Reproduced with the permission of the Controller of Her Majesty's Stationery Office/Queen's Printer for Scotland and AHVLA.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

PubMed Central

2012-01-01

Background Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). Results Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. Conclusions Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies. PMID:22857656

[Assessment of cervical intraepithelial neoplasia (CIN) lesions by DNA image cytometry].

PubMed

Sun, Xiao-rong; Che, Dong-yuan; Tu, Hong-zhang; Li, Dan; Wang, Jian

2006-11-01

To compare the value of conventional cytology and DNA image cytometry (DNA-ICM) assisted cytology in detection and prognostic assessment of cervical CIN lesions. 87 women were enrolled in this study. Cervical samples were collected employing cervix brushes which were then washed in Sedfix. After preparing single cell suspensions by mechanical procedure, cell monolayers were prepared by cyto-spinning the cells onto microscope slides. Two slides were prepared from each case: one slide was stained by Papanicolou staining for conventional cytology, another was stained by Feulgen-Thionin method for measurements of the amount of DNA in the cell nuclei using an automated DNA imaging cytometer. Biopsies from the cervical lesions were also taken for histopathology and Ki-67 immunohistochemistry. Of the total of 20 ASCUS cases called by conventional cytology, no CIN, nor greater lesions were found. Among the 20 cases, 7 cases did not show any cells with DNA amount greater than 5c, while CIN2 lesions were found in 11 of other 13 cases that had some aneuploid cells with DNA amount greater than 5c. Of 30 LSIL cases called by conventional cytology, CIN2 lesions were detected in 3 out of 7 cases that did not contain any aneuploid cells with DNA greater than 5c, but in 22 out of the other 23 cases that contained aneuploid cells with DNA amount greater than > 5c. Of the remaining 7 cases called HSIL by conventional cytology, all case contained aneuploid cells containing DNA greater than 5c. If cytology was used to refer all cases of LSIL and HSIL to colposcopy procedure to detect potential CIN2 or greater lesions, the sensitivity, specificity, positive predictive value and negative predictive value were 58.2%, 84.4%, 86.5% and 54.0%, respectively. If DNA-ICM were used and all cases having 3 or more cells with a DNA amount greater than 5c were assessed to be referred to pathology to detect potential CIN2 or greater lesions, the sensitivity, specificity, positive predictive value and negative predictive were 72.7% , 87.5%, 90.9% and 65.1%, respectively. We also compared Ki67 positive cells in these samples and found that DNA-ICM results were comparable to this biomarker method. The study demonstrated that DNA-ICM approach can be successfully used to detect significant (i.e. CIN2 or greater) lesions, and also provide a prognostic assessment of CIN lesions.

Observation of the density and size of cells in hippocampus and vascular lesion in thalamus of GFAP-apoE transgenic mice.

PubMed

Tang, Ke-Feng; Cai, Li; Zhou, Jiang-Ning

2009-08-01

Apolipoprotein E (apoE) is associated with increased risk of age-related diseases, such as Alzheimer's disease (AD) and cerebrovascular disease (CVD). The present study aims to investigate the age-related general morphological changes of the brain in GFAP-apoE transgenic mice, especially the alterations in number and size of hippocampal pyramidal cells and the microvascular lesions in the thalamus. Nine female apoE4/4 mice were divided into 3 groups (n=3 in each group): 3-4 months (young group), 9-10 months (middle-aged group) and 20-21 months (old group). Age-matched apoE3/3 mice were employed as control group (n=3 in each group). The paraffin sections of brain tissue were stained by 2 conventional staining methods, thionin staining and hematoxylin-esion(HE) staining, the former of which was to observe the hippocampal cells, while the latter was used to examine the brain microvasculature. There was no apparent difference in the cortical layer between apoE3/3 and apoE4/4 mice, neither any significant difference in the number of cells in hippocampal CA1-CA3 subfields between apoE3/3 and apoE4/4 mice at various age points (P>0.05). However, the mean size of pyramidal cells in CA1 subfield in apoE3/3 and apoE4/4 mice decreased as mice were getting older (P<0.001). At the age of 20-21 months, this cellular atrophy in apoE4/4 mice was more severe than that in old apoE3/3 mice (P<0.05). Furthermore, microvascular lesion in the thalamus was detected in all the 3 old apoE4/4 mice, at varying degrees (5.24%, 1.41% and 3.97%, respectively), while only one apoE3/3 mouse exhibited microvascular lesion in the thalamus, at a low level (0.85%). The current study suggests that the cell size in hippocampal CA1 subfield decreases with aging, irrespective of apoE genotype. Cellular atrophy in CA1 subfield and the microvascular lesion in the thalamus are both more severe in old apoE4/4 mice as compared with those in age-matched apoE3/3 mice. Doubts still exist on whether the decreased cell size in hippocampal CA1 subfield in old apoE4/4 mice is associated with dysfunction in learning and memory and whether the microvascular lesions indicate a higher risk of stroke in human apoE4 allele mice. To clarify these issues, further investigations are needed.

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata.

PubMed

Dracatos, Peter M; van der Weerden, Nicole L; Carroll, Kate T; Johnson, Elizabeth D; Plummer, Kim M; Anderson, Marilyn A

2014-01-01

Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens. © 2013 BSPP AND JOHN WILEY & SONS LTD.