scholarly journals Development of a Robust Screening Method for Pathogenicity of Colletotrichum spp. on Strawberry Seedlings Enabling Forward Genetic Studies

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 845-851 ◽  
Author(s):  
Sigal Horowitz ◽  
Oded Yarden ◽  
Aida Zveibil ◽  
Stanley Freeman
Keyword(s):  
Large Scale ◽  
Developmental Stages ◽  
Screening Method ◽  
Rapid Screening ◽  
Disease Symptoms ◽  
Inoculation Techniques ◽  
Lower Temperature ◽  
Incubation Temperatures

Generation and screening for nonpathogenic mutants is a popular tool for identifying pathogenicity-related genes. Successful application of this technique for plant fungal pathosystems requires reliable and rapid screening procedures. This study reports on the development of a rapid in vitro bioassay enabling large-scale screening and isolation of nonpathogenic mutants of Colletotrichum gloeosporioides and C. acutatum on strawberry seedlings. Inoculation was carried out on strawberry seedlings at two different developmental stages: 12-week-old (young) and 15-week-old (older) seedlings. A comparison was made between two inoculation techniques, (i) foliar dip and (ii) root soak, at two incubation temperatures (19 and 25°C). Mortality of young seedlings was observed 4 days after inoculation with both species, reaching 50% within 10 days, using both techniques at 25°C. However, mortality of older seedlings was delayed by 4 days compared with that in the young seedlings when using the root-soak method. Disease development decreased in young and older seedlings at the lower temperature. This method also was reliable in determining pathogenicity of the cucurbit-specific C. magna that did not cause disease symptoms on strawberry by either inoculation method. The proposed method enabled screening of more than 980 restriction enzyme-mediated integration mutants resulting in a selection of five reduced-virulence isolates. Initial characterization of some of these mutants revealed large differences in germination and appressorial formation compared with pathogenic isolates.

scholarly journals Phytochemical Profiles, Physicochemical Analysis, and Biological Activity of Three Colchicum Species

2021 ◽  
Vol 16 (2) ◽  
Author(s):  
Ali Davoodi ◽  
Mohammad Azadbakht ◽  
Seyed Jalal Hosseinimehr ◽  
Saeed Emami ◽  
Masoud Azadbakht
Keyword(s):  
Physicochemical Properties ◽  
Large Scale ◽  
Protein Denaturation ◽  
Potassium Dichromate ◽  
Biological Effects ◽  
Screening Method ◽  
Rapid Screening ◽  
Total Phenolic ◽  
Anti Inflammatory

Background: Colchicum is a genus of the Colchicaceae family with various isolated compounds, especially tropolone alkaloids and flavonoids, which are used for osteoarthritis, gout, cancer, inflammatory diseases, jaundice, and sexual impotence in different societies. Objectives: The current study aimed to evaluate the phytochemical and physicochemical properties and anti-inflammatory activities of three Colchicum species. Methods: Total tropolone alkaloid, total phenolic/total tannin, and total flavonoid contents were determined using acidic potassium dichromate, Folin-Ciocalteu, and aluminum chloride methods, respectively. Moreover, the HPLC method was used for identification and quantitation purposes of tropolone alkaloids. Physicochemical properties of three Colchicum species, including macroscopic and organoleptic properties, solubility, foreign matter, ash values, and heavy metal contents, were evaluated. Besides, in vitro anti-inflammatory activities of the corms also were determined using the protein denaturation technique as a rapid screening method. Results: The highest levels of tropolone alkaloid, phenolic compounds, tannins, and flavonoids were observed in C. autumnale, C. speciosum, and C. robustum, respectively. HPLC analysis indicated the presence of colchicine, demecolcine, 2-demethyl colchicine, 3-demethyl colchicine, colchicoside, colchifoline, cornigerine, and N-deacetyl-N-formyl colchicine in these Colchicum species. The physicochemical properties of C. speciosum and C. robustum corms are appropriate compared to the standard Colchicum autumnale corm. Moreover, all Colchicum species exhibited high anti-inflammatory activities compared to standard drugs. Conclusions: This study demonstrated that the corm of the Colchicum species contained similar main compounds with different amounts, as well as appropriate physicochemical properties. Moreover, the valuable biological effects of these plants stimulate the cultivations on a large scale.

scholarly journals Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

2005 ◽  
Vol 73 (2) ◽  
pp. 972-980 ◽  
Author(s):  
C. G. Osorio ◽  
J. A. Crawford ◽  
J. Michalski ◽  
H. Martinez-Wilson ◽  
J. B. Kaper ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Large Scale ◽  
Screening Method ◽  
Mouse Small Intestine ◽  
In Vivo Expression ◽  
Induced Genes ◽  
El Tor ◽  
Selection Of

ABSTRACT We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages

Parasitology ◽  
2014 ◽  
Vol 142 (04) ◽  
pp. 598-611 ◽  
Author(s):  
PETER H. GAUDET ◽  
RICHARD J. CAWTHORN ◽  
MELANIE A. BUOTE ◽  
J. FRANK MORADO ◽  
GLENDA M. WRIGHT ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Partial Characterization ◽  
Snow Crab ◽  
In Vitro Cultivation ◽  
Chionoecetes Opilio

scholarly journals From Zero to Neuro-Reprogramming: Innovations in Translational Neuroregenerative Medicine

2019 ◽  
Vol 9 (1) ◽  
pp. 54-60
Author(s):  
Ahmad Galuta ◽  
Eve Tsai
Keyword(s):  
Stem Cells ◽  
Nervous System ◽  
Large Scale ◽  
Neuronal Development ◽  
Developmental Stages ◽  
Study Patient ◽  
Patient Specific ◽  
Translational Study ◽  
Neuroregenerative Medicine

Acquiring live human nervous tissue for research presents ethical and technical constraints. As a result, clinicians and scientists resort to using animal models to investigate human neuronal development and degeneration. However, innate species differences in neurobiology have hindered the translation of disease pathologies and development of therapeutic strategies. The discovery of endogenous neural stem cells (NSCs) and their examination has been critical for neuronal development, degeneration and regeneration. NSCs can exist in different developmental stages, embryonic through adult, and possess the capacity to generate the various cells that make up the nervous system. Importantly, human somatic cells can be obtained non-invasively and genetically reprogrammed into NSCs providing an ethically viable source of stem cells for translational study and potential therapy. Novel methods to generate NSCs of various developmental origins and regional identities are rapidly evolving to provide safer, quicker, and more efficient reprogramming strategies. Reprogrammed NSCs share many molecular and functional attributes with their endogenous NSC counterparts and can be used for in vitro modelling at a large scale. The accessibility to study patient specific NSCs allows the causal inferences of human disease mechanisms that may be unfeasible to model in animals. Despite the novelty of this burgeoning field, the opportunity for translational discoveries in neuronal development and degeneration and for therapeutic applications is unprecedented. This review will highlight the advances in manufacturing NSCs and their translational implications for disease modelling and potential treatment of the nervous system.

scholarly journals MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.)

2016 ◽  
Vol 8 (2) ◽  
pp. 43 ◽  
Author(s):  
Pauline D. Kasi ◽  
Sumaryono Sumaryono
Keyword(s):  
Developmental Stage ◽  
Somatic Embryos ◽  
Large Scale ◽  
Developmental Stages ◽  
Morphological Changes ◽  
Petri Dish ◽  
Synthetic Seed ◽  
Globular Embryos ◽  
Metroxylon Sagu

Development of somatic embryos of sago (Metroxylon sagu Rottb.) on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sago<br />somatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish) were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS) with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8%) and reddish (95.8%) embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%<br />remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%). Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

scholarly journals Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Suwen Zhao ◽  
Ayano Sakai ◽  
Xinshuai Zhang ◽  
Matthew W Vetting ◽  
Ritesh Kumar ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Large Scale ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Enzymatic Activities ◽  
Or Gene ◽  
Enzyme Functions

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

scholarly journals CloneSifter: enrichment of rare clones from heterogeneous cell populations

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
David Feldman ◽  
FuNien Tsai ◽  
Anthony J. Garrity ◽  
Ryan O’Rourke ◽  
Lisa Brenan ◽  
...  
Keyword(s):  
Large Scale ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Population Level ◽  
Fitness Assessment ◽  
Large Populations ◽  
Organismal Development ◽  
Insight Into

Abstract Background Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones. Results Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population. Conclusions CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.

scholarly journals Chemical Characterization of Narcissus poeticus from Sirente –Velino (Apennines - Italy): Galantamine Accumulation and Distribution of Allergenic Compounds in the Flower

2017 ◽  
Vol 12 (1) ◽  
pp. 1934578X1701200
Author(s):  
Donatella Ferri ◽  
Carla Ubaldi ◽  
Giordana Marcozzi ◽  
Paolo Fasciani ◽  
Loretta Bacchetta ◽  
...  
Keyword(s):  
In Vitro Propagation ◽  
Large Scale ◽  
Chemical Characterization ◽  
Potential Source ◽  
Flower Stem ◽  
Narcissus Poeticus ◽  
Extraction Solvents ◽  
Plant Flower

Species of Narcissus (family Amaryllidaceae) are a potential source for large-scale extraction of alkaloids and fragrances. The bulbs typically accumulate a large number of alkaloids, including galantamine, a benzazepine alkaloid proven to be a cholinesterase inhibitor and which is used in the treatment of Alzheimer's disease. The presence of galantamine in N. poeticus L. collected in Abruzzo (Italy) was assessed and several levels of alkaloid were found in all parts of the plant (flower, stem, bulb and root) and not only in the bulb. The amount of galantamine obtained was tested by using two different extraction solvents. Extraction of N. poeticus absolute from the flowers was also performed, as this product is an important floral note in perfumery, and the distribution of allergenic compounds in the coronas and in the tepals was assessed. Moreover, the in vitro propagation of N. poeticus was tested as it may be a valuable resource from which to produce biomolecules, as an alternative to chemical synthetic processes.

scholarly journals Hydropathicity-based prediction of pain-causing NaV1.7 variants

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Makros N. Xenakis ◽  
Dimos Kapetis ◽  
Yang Yang ◽  
Monique M. Gerrits ◽  
Jordi Heijman ◽  
...  
Keyword(s):  
Computational Cost ◽  
Clinical Importance ◽  
Operating Characteristics ◽  
Functional Architecture ◽  
Disease Symptoms ◽  
Receiver Operating Characteristics Curve ◽  
Reducing Costs ◽  
Hydropathic Interactions

Abstract Background Mutation-induced variations in the functional architecture of the NaV1.7 channel protein are causally related to a broad spectrum of human pain disorders. Predicting in silico the phenotype of NaV1.7 variant is of major clinical importance; it can aid in reducing costs of in vitro pathophysiological characterization of NaV1.7 variants, as well as, in the design of drug agents for counteracting pain-disease symptoms. Results In this work, we utilize spatial complexity of hydropathic effects toward predicting which NaV1.7 variants cause pain (and which are neutral) based on the location of corresponding mutation sites within the NaV1.7 structure. For that, we analyze topological and scaling hydropathic characteristics of the atomic environment around NaV1.7’s pore and probe their spatial correlation with mutation sites. We show that pain-related mutation sites occupy structural locations in proximity to a hydrophobic patch lining the pore while clustering at a critical hydropathic-interactions distance from the selectivity filter (SF). Taken together, these observations can differentiate pain-related NaV1.7 variants from neutral ones, i.e., NaV1.7 variants not causing pain disease, with 80.5$$\%$$ % sensitivity and 93.7$$\%$$ % specificity [area under the receiver operating characteristics curve = 0.872]. Conclusions Our findings suggest that maintaining hydrophobic NaV1.7 interior intact, as well as, a finely-tuned (dictated by hydropathic interactions) distance from the SF might be necessary molecular conditions for physiological NaV1.7 functioning. The main advantage for using the presented predictive scheme is its negligible computational cost, as well as, hydropathicity-based biophysical rationalization.

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