Laboratory Protocol for Induction of Sporulation in Phytophthora cajani causing Phytophthora Blight in Pigeonpea

2020 ◽  
Author(s):  
G. Jadesha ◽  
Mamta Sharma ◽  
P. Narayan Reddy
Keyword(s):  
Incubation Period ◽  
Culture Conditions ◽  
Fluorescent Light ◽  
Limited Information ◽  
Phytophthora Blight ◽  
Phytophthora Spp ◽  
Tomato Extract ◽  
Culture Plate ◽  
Laboratory Protocol

Background: Phytophthora blight (PB), caused by Phytophthora cajani is a prominent disease in the low laying areas combined with intermittent rainfall. Induction of zoospores and sporangia of P. cajani in culture plate is troublesome and also limited information is available on the protocol for sporulation of sporangia and zoospores in the laboratory, The study developed and validated the protocol for induction of sporangia and zoospore of P. cajani. Methods: The Protocol using 5-7 days old culture, diluted tomato extract broth and other culture conditions like the temperature of 30oC, alternate 12 hours of fluorescent light (2000 Lx) and dark can induce abundant sporangia and zoospores in vitro. Result: The study developed and validated the new protocol for uniform and profuse induction of sporangia and zoospores of P. cajani. Diplanetism mechanism of Phytophthora spp. was recorded with P. cajani the findings are the first of its kind. Additionally, the study revealed the concentration of 1x10-5 zoospores/ml is optimum to develop the infection in plants with the shortest incubation period of 24 hours.

Illustration of Key Morphological Characteristics of Phytophthora cajani-Pathogen of Phytophthora Blight of Pigeonpea

2020 ◽  
Author(s):  
G. Jadesha ◽  
Mamta Sharma ◽  
P. Narayan Reddy
Keyword(s):  
Phase Contrast ◽  
Morphological Characteristics ◽  
Phytophthora Blight ◽  
Phytophthora Spp ◽  
Cropping Season ◽  
Contrast Microscope ◽  
Culture Characteristics ◽  
Stimulation Of

Background: Phytophthora cajani causing the Phytophthora blight (PB) disease of pigeonpea. The disease will rampant during excessive rainfall coupled with hot and humid weather during the cropping season. The present study on micro and macro morphological characteristics can contribute to the identification and specification of biology of Phytophthora spp. There are no detailed studies concerning the characterization of the P. cajani are available with this backdrop the present investigation was taken. Methods: Phytophthora cajani was isolated on V-8 PARP medium, whereas stimulation of zoospores and sporangia was done using the diluted tomato juice broth. Micro and macro morphological characteristics of P. cajani were studied using micrometry and Olympus CX41 phase-contrast microscope. Result: The pathogen was homothallic with amphigynous antheridium and oogonium and able to produce oospore in vitro. Sporangium was nonpapillate, noncaducous, oviod-obpyriform shape. Further, the macro morphological characteristics like mycelial radial growth and colony type were studied. The colony characteristics were dull white, flat and rosette pattern. Other culture characteristics like optimum temperature and RH were mostly consistent with those reported former.

scholarly journals EFFECT OF CULTURE CONDITIONS FOR ANTIMICROBIAL ACTIVITY OF MARINE - DERIVED FUNGUS ASPERGILLUS FLOCCULOSUS 01NT.1.1.5

2018 ◽  
Vol 15 (4) ◽  
pp. 721-728
Author(s):  
Phan Thi Hoai Trinh ◽  
Ngo Thi Duy Ngoc ◽  
Vo Thi Dieu Trang ◽  
Phi Quyet Tien ◽  
Bui Minh Ly ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Secondary Metabolites ◽  
Incubation Period ◽  
Marine Fungi ◽  
Antibiotic Production ◽  
Morphological Characteristics ◽  
Colony Growth ◽  
Culture Conditions ◽  
Initial Ph

The biosynthesis of compounds with antibiotic activity produced by marine fungi, strongly depends on their growth conditions. A good understanding of the role of culture conditions in the biosynthesis of metabolites may lead to better exploitation of microbial metabolites. In this study, the influence of culture conditions including incubation period, initial pH and salinity on antimicrobial activity and secondary metabolites production of marine fungus 01NT.1.1.5 was investigated. This isolate, obtained from sponge Stylissa sp. in Nha Trang Bay, exhibited a broad spectrum of in vitro antimicrobial activity to Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111, Streptococcus faecalis ATCC 19433 and Candida albicans ATCC 10231. According to morphological characteristics and sequence analysis of 28S rDNA, the fungus was identified as Aspergillus flocculosus. The results indicated that antimicrobial activity and metabolite amount were highest when the fungus was cultivated in rice medium with incubation period of 20 days. The optimum salinity of 35 g/L and initial pH of 6.0 were found for the maximum antibiotic production. The colony growth, antimicrobial activity and production of secondary metabolites of the strain A. flocculosus 01NT.1.1.5 varied depending on salt concentrations and initial pH of medium. Particularly, extract of this fungus only showed activity against C. albicans when it was cultured in medium with 30-35 g/L salinity and initial pH 4.0-8.0. The results  indicate that salinity and initial pH along with cultivation period are important factors influencing antimicrobial activity and secondary metabolites of A. flocculosus 01NT.1.1.5, and might be for other marine fungi.

151 COMPARISON OF CELL NUMBERS OF ZONA-INTACT AND ZONA-FREE PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS CULTURED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 234 ◽  
Author(s):  
J. Li ◽  
G. Vjata ◽  
H. Callesen
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida ◽  
Cumulus Cell ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Fluorescent Light ◽  
Average Cell ◽  
Cell Numbers

Application of an artificial stimulus to activate oocytes and induce development is essential for the successful animal cloning by nuclear transfer (Zhu et al. 2002 Biol. Reprod. 66, 635-641). The embryo’s developmental competence could be further improved with optimal in vitro culture conditions (Du et al. 2007 Theriogenology 68, 1104-1110). Cell number determination is a commonly used and simple criterion to assess developmental quality of pre-implantation stage mammalian embryos (Lagutina et al. 2007 Theriogenology 67, 90-98). Our aim of the study was to investigate porcine embryos activated and cultured in different ways using total cell numbers as the only quality measure. After 43-44 h of in vitro maturation and cumulus cell removal, zona-intact (PAZI) or zona-free oocytes (PAZF; after pronase treatment) were subjected to parthenogenetic activation (Day 0) with a single 80-μs DC pulse of 1.26 kV cm-1 or 0.86 kV cm-1 (Kragh et al. 2005 Theriogenology 64, 1536-1545), followed by a 4-h treatment with 5 μg mL-1 of cytochalasin B and 10 μg mL-1 of cycloheximide. Subsequently, the well of the well system (Vajta in vitro 2000 Mol. Reprod. Dev. 55, 256-264) was used for culture of all PAZF and half of the PAZI embryos (PAZF-WOW and PAZI-WOW groups, respectively), whereas the remaining PAZI embryos were cultured in groups of 25-30 (PAZI group). All cultures were performed in porcine zygote medium 3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119). On Day 6, all these in vitro cultured embryos were fixed and stained with Hoechst 33342 and cell numbers were counted on the microscopic pictures taken using fluorescent light. Data analysis was performed using ANOVA. Four replicates were performed with a total of 462 PAZF-WOW, 484 PAZI-WOW, and 467 PAZI group embryos. Embryos of each group were then divided into 5 groups based on their cell number (<5 cells, 5-8 cells, 9-16 cells, 17-32 cells, >32 cells). Percentages of embryos in each group are shown in Table 1. The average cell numbers of zona-intact embryos from PAZI-WOW and the PAZI group were similar to each other (P > 0.05), whereas the cell numbers of PAZF-WOW embryos were significantly different from both PAZI-WOW and PAZI group embryos (P < 0.05), with more embryos having higher cell numbers.The results demonstrate that zona-free parthenogenetically activated embryos cultured in WOW have higher cell numbers than embryos with intact zona pellucida. Accordingly, the presence of zona pellucida may compromise embryo development under certain in vitro culture situations. Table 1.Distribution (in %) of parthenogenetically activated porcine embryos according to their cell number on Day 6 after activation

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Effect of Temperature on ADP-Induced Platelet Aggregation

1973 ◽  
Vol 29 (01) ◽  
pp. 183-189
Author(s):  
C. A Praga ◽  
E. M Pogliani
Keyword(s):  
Platelet Aggregation ◽  
Incubation Period ◽  
Room Temperature ◽  
Important Variable ◽  
Practical Significance ◽  
Effect Of Temperature ◽  
Induce Platelet Aggregation ◽  
Cuvette Holder ◽  
Exact Temperature

SummaryTemperature represents a very important variable in ADP-induced platelet aggregation.When low doses of ADP ( < 1 (μM) are used to induce platelet aggregation, the length of the incubation period of PRP in the cuvette holder of the aggregometer, thermostatted at 37° C, is very critical. Samples of the same PRP previously kept at room temperature, were incubated for increasing periods of time in the cuvette of the aggregometer before adding ADP, and a significant decrease of aggregation, proportional to the length of incubation, was observed. Stirring of the PRP during the incubation period made these changes more evident.To measure the exact temperature of the PRP during incubation in the aggre- gometer, a thermocouple device was used. While the temperature of the cuvette holder was stable at 37° C, the PRP temperature itself increased exponentially, taking about ten minutes from the beginning of the incubation to reach the value of 37° C. The above results have a practical significance in the reproducibility of the platelet aggregation test in vitro and acquire particular value when the effect of inhibitors of ADP induced platelet aggregation is studied.Experiments carried out with three anti-aggregating agents (acetyl salicyclic acid, dipyridamole and metergoline) have shown that the incubation conditions which influence both the effect of the drugs on platelets and the ADP breakdown in plasma must be strictly controlled.

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

scholarly journals The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 360
Author(s):  
Pieterjan Debie ◽  
Noemi B. Declerck ◽  
Danny van Willigen ◽  
Celine M. Huygen ◽  
Bieke De Sloovere ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gamma Ray ◽  
Nuclear Imaging ◽  
Primary Amines ◽  
Resection Margins ◽  
Fluorescent Light ◽  
Single Structure ◽  
Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

scholarly journals Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Renata Orłowska
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Taguchi Method ◽  
Culture Conditions ◽  
Dna Demethylation ◽  
Silver Ion ◽  
Donor Plant ◽  
In Vitro Tissue Culture ◽  
Induced Variation

Abstract Background Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. Results This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Conclusions The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

Sign in / Sign up

Export Citation Format

Share Document