scholarly journals Soil Bioassay for Detecting Magnaporthiopsis maydis Infestation Using a Hyper Susceptible Maize Hybrid

2020 ◽  
Vol 6 (3) ◽  
pp. 107
Author(s):  
Ofir Degani ◽  
Danielle Regev ◽  
Shlomit Dor ◽  
Onn Rabinovitz
Keyword(s):  
Vascular System ◽  
High Sensitivity ◽  
Development Stage ◽  
Plant Residues ◽  
Potted Plants ◽  
Soil Bioassay ◽  
Maize Genotype ◽  
Qpcr Method ◽  
Infested Field

Magnaporthiopsis maydis is the causal agent of severe maize late wilt disease. Disease outbreak occurs at the maize flowering and fruit development stage, leading to the plugging of the plant’s water vascular system, resulting in dehydration and collapse of the infected host plant. The pathogen is borne by alternative hosts, infected seeds, soil, and plant residues and gradually spreads to new areas and new countries. However, no soil assay is available today that can detect M. maydis infestation and study its prevalence. We recently developed a molecular quantitative Real-Time PCR (qPCR) method enabling the detection of the M. maydis DNA in plant tissues. Despite the technique’s high sensitivity, the direct examination of soil samples can be inconsistent. To face this challenge, the current work demonstrates the use of a soil bioassay involving the cultivation of a hyper-susceptible maize genotype (Megaton cultivar, Hazera Seeds Ltd., Berurim MP Shikmim, Israel) on inspected soils. The use of Megaton cv. may facilitate pathogen establishment and spread inside the plant’s tissues, and ease the isolation and enrichment of the pathogen from the soil. Indeed, this cultivar suffers from severe dehydration sudden death when grown in an infested field. The qPCR method was able to accurately and consistently identify and quantify the pathogen’s DNA in an in vitro seed assay after seven days, and in growth-chamber potted plants at as early as three weeks. These results now enable the use of this highly susceptible testing plant to validate the presence of the maize late wilt pathogen in infested soils and to evaluate the degree of its prevalence.

scholarly journals Validation of the in vitro comet assay for DNA cross-links and altered bases detection

2021 ◽  
Author(s):  
Damián Muruzabal ◽  
Julen Sanz-Serrano ◽  
Sylvie Sauvaigo ◽  
Bertrand Treillard ◽  
Ann-Karin Olsen ◽  
...  
Keyword(s):  
Comet Assay ◽  
Human Health Risk ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Vitro Assay ◽  
Cytotoxic Compound ◽  
Endonuclease Iii ◽  
Cross Links

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

Independent and combined effects of biotin and hemolysis on high-sensitivity cardiac troponin assays

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Kellisha Harley ◽  
Sarah Bissonnette ◽  
Rosanna Inzitari ◽  
Karen Schulz ◽  
Fred S. Apple ◽  
...  
Keyword(s):  
Cardiac Troponin ◽  
Clinical Laboratory ◽  
Troponin T ◽  
High Sensitivity ◽  
Combined Effects ◽  
Over The Counter ◽  
Heparin Plasma ◽  
Abbott Architect ◽  
Roche Cobas

Abstract Objectives This study compared the independent and combined effects of hemolysis and biotin on cardiac troponin measurements across nine high-sensitivity cardiac troponin (hs-cTn) assays. Methods Parallel cTn measurements were made in pooled lithium heparin plasma spiked with hemolysate and/or biotin using nine hs-cTn assays: Abbott Alinity, Abbott ARCHITECT i2000, Beckman Access 2, Ortho VITROS XT 7600, Siemens Atellica, Siemens Centaur, Siemens Dimension EXL cTnI, and two Roche Cobas e 411 Elecsys Troponin T-hs cTnT assays (outside US versions, with and without increased biotin tolerance). Absolute and percent cTn recovery relative to two baseline concentrations were determined in spiked samples and compared to manufacturer’s claims. Results All assays except the Ortho VITROS XT 7600 showed hemolysis and biotin interference thresholds equivalent to or greater than manufacturer’s claims. While imprecision confounded analysis of Ortho VITROS XT 7600 data, evidence of biotin interference was lacking. Increasing biotin concentration led to decreasing cTn recovery in three assays, specifically both Roche Cobas e 411 Elecsys Troponin T-hs assays and the Siemens Dimension EXL. While one of the Roche assays was the most susceptible to biotin among the nine studied, a new version showed reduced biotin interference by approximately 100-fold compared to its predecessor. Increasing hemolysis also generally led to decreasing cTn recovery for susceptible assays, specifically the Beckman Access 2, Ortho VITROS XT 7600, and both Roche Cobas e 411 Elecsys assays. Equivalent biotin and hemolysis interference thresholds were observed at the two cTn concentrations considered for all but two assays (Beckman Access 2 and Ortho VITROS XT 7600). When biotin and hemolysis were present in combination, biotin interference thresholds decreased with increasing hemolysis for two susceptible assays (Roche Cobas e 411 Elecsys and Siemens Dimension EXL). Conclusions Both Roche Cobas e 411 Elecsys as well as Ortho VITROS XT assays were susceptible to interference from in vitro hemolysis at levels routinely encountered in clinical laboratory samples (0–3 g/L free hemoglobin), leading to falsely low cTn recovery up to 3 ng/L or 13%. While most assays are not susceptible to biotin at levels expected with over-the-counter supplementation, severely reduced cTn recovery is possible at biotin levels of 10–2000 ng/mL (41–8,180 nmol/L) for some assays. Due to potential additive effects, analytical interferences should not be considered in isolation.

scholarly journals The cancer glycocalyx mediates intravascular adhesion and extravasation during metastatic dissemination

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Giovanni S. Offeddu ◽  
Cynthia Hajal ◽  
Colleen R. Foley ◽  
Zhengpeng Wan ◽  
Lina Ibrahim ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Tumor Cells ◽  
Cancer Progression ◽  
Vascular System ◽  
Migration And Invasion ◽  
Improve Patient Survival ◽  
Adhesive Interactions ◽  
Vitro Model ◽  
Metastatic Sites

AbstractThe glycocalyx on tumor cells has been recently identified as an important driver for cancer progression, possibly providing critical opportunities for treatment. Metastasis, in particular, is often the limiting step in the survival to cancer, yet our understanding of how tumor cells escape the vascular system to initiate metastatic sites remains limited. Using an in vitro model of the human microvasculature, we assess here the importance of the tumor and vascular glycocalyces during tumor cell extravasation. Through selective manipulation of individual components of the glycocalyx, we reveal a mechanism whereby tumor cells prepare an adhesive vascular niche by depositing components of the glycocalyx along the endothelium. Accumulated hyaluronic acid shed by tumor cells subsequently mediates adhesion to the endothelium via the glycoprotein CD44. Trans-endothelial migration and invasion into the stroma occurs through binding of the isoform CD44v to components of the sub-endothelial extra-cellular matrix. Targeting of the hyaluronic acid-CD44 glycocalyx complex results in significant reduction in the extravasation of tumor cells. These studies provide evidence of tumor cells repurposing the glycocalyx to promote adhesive interactions leading to cancer progression. Such glycocalyx-mediated mechanisms may be therapeutically targeted to hinder metastasis and improve patient survival.

Healthy and diseased in vitro models of vascular systems

Lab on a Chip ◽  
2021 ◽  
Vol 21 (4) ◽  
pp. 641-659
Author(s):  
Vahid Hosseini ◽  
Anna Mallone ◽  
Fatemeh Nasrollahi ◽  
Serge Ostrovidov ◽  
Rohollah Nasiri ◽  
...  
Keyword(s):  
Critical Review ◽  
Vascular System ◽  
In Vitro Models

A critical review of healthy and diseased in vitro models of the vascular system and in particular for atherosclerosis, aneurysm, and thrombosis.

scholarly journals X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples

2021 ◽  
Vol 22 (7) ◽  
pp. 3691
Author(s):  
Oliver Schmutzler ◽  
Sebastian Graf ◽  
Nils Behm ◽  
Wael Y. Mansour ◽  
Florian Blumendorf ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Irradiation Time ◽  
High Sensitivity ◽  
Nanoparticle Uptake ◽  
X Ray ◽  
Functionalized Gold Nanoparticles ◽  
Uptake Measurement ◽  
Prostate Tumor Cells ◽  
Non Destructive

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2157-2169 ◽  
Author(s):  
Sudarson Sundarrajan ◽  
Junjappa Raghupatil ◽  
Aradhana Vipra ◽  
Nagalakshmi Narasimhaswamy ◽  
Sanjeev Saravanan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanism Of Action ◽  
Catalytic Domain ◽  
Antibiotic Sensitivity ◽  
High Sensitivity ◽  
Common Mechanism ◽  
Cross Bridge ◽  
Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

Compressive Force Induces VEGF Production in Periodontal Tissues

2009 ◽  
Vol 88 (8) ◽  
pp. 752-756 ◽  
Author(s):  
A. Miyagawa ◽  
M. Chiba ◽  
H. Hayashi ◽  
K. Igarashi
Keyword(s):  
Alveolar Bone ◽  
Tooth Movement ◽  
Vascular System ◽  
Orthodontic Tooth Movement ◽  
Compressive Force ◽  
Vegf Mrna ◽  
Angiogenic Activity ◽  
Periodontal Tissues ◽  
Pdl Cells

During orthodontic tooth movement, the activation of the vascular system in the compressed periodontal ligament (PDL) is an indispensable process in tissue remodeling. We hypothesized that compressive force would induce angiogenesis of PDL through the production of vascular endothelial growth factor (VEGF). We examined the localization of VEGF in rat periodontal tissues during experimental tooth movement in vivo, and the effects of continuous compressive force on VEGF production and angiogenic activity in human PDL cells in vitro. PDL cells adjacent to hyalinized tissue and alveolar bone on the compressive side showed marked VEGF immunoreactivity. VEGF mRNA expression and production in PDL cells increased, and conditioned medium stimulated tube formation. These results indicate that continuous compressive force enhances VEGF production and angiogenic activity in PDL cells, which may contribute to periodontal remodeling, including angiogenesis, during orthodontic tooth movement.

scholarly journals Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

2003 ◽  
Vol 93 (8) ◽  
pp. 953-958 ◽  
Author(s):  
W.-B. Li ◽  
W. D. Pria ◽  
P. M. Lacava ◽  
X. Qin ◽  
J. S. Hartung
Keyword(s):  
Vascular System ◽  
Sweet Orange ◽  
Xylella Fastidiosa ◽  
Germination Rate ◽  
Citrus Fruit ◽  
Vascular Bundles ◽  
Specific Amplification ◽  
Root Grafts ◽  
Citrus Seeds

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

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