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Environmental Entomology Feb 2023Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana...
Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana can act as semiochemical attractants or repellents, with most responses reported to date resulting in insects avoiding B. bassiana. Since insects can detect 'enemy-specific volatile compounds', we hypothesized the preference behavior of M. persicae would be influenced by volatile emissions from B. bassiana. We conducted Petri dish and Y-tube olfactometer bioassays to characterize the preference of M. persicae to B. bassiana strain GHA. During Petri dish bioassays, more apterous and alate M. persicae were recorded in the vicinity of agar colonized by B. bassiana compared to agar, or Fusarium proliferatum (Matsushima) Nirenberg and Ambrosiella grosmanniae Mayers, McNew, & Harrington as representatives of nonentomopathogenic fungi. Petri dish bioassays also determined that apterous and alate M. persicae preferred filter paper saturated with 1 × 107, 1 × 106, and 1 × 105B. bassiana conidia/ml compared to Tween 80. Y-tube bioassays documented that more apterous and alate M. persicae oriented upwind to volatiles from B. bassiana mycelia compared to agar. Apterous and alate Myzus persicae were also preferentially attracted to 1 × 107 and 1 × 106B. bassiana conidia/ml compared to Tween-80 during Y-tube bioassays. These results complement a previous finding that the mosquito Anopheles stephensi (Diptera: Culicidae) Liston is attracted to volatiles from B. bassiana. Future studies aimed at characterizing the olfactory mechanism leading to the attraction of M. persicae to B. bassiana could aid in optimizing lure-and-kill strategies.
Topics: Animals; Beauveria; Aphids; Agar; Spores, Fungal; Pest Control, Biological
PubMed: 36421055
DOI: 10.1093/ee/nvac100 -
Materials (Basel, Switzerland) Aug 2019The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials...
The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials are shown to affect the wound-healing process in a cell-type dependent manner. However, experiment-to-experiment variations make it difficult to compare results from different assays. In this paper, a modified barrier wound-healing assay was reported for studying the wound-healing process on different substrates in one single petri dish. In short, half of a dish was covered with the tape, and coating materials, poly-l-lysine and gelatin, were applied to the surface. After peeling off the tape, half of the surface was coated with the desired material. Then a customized barrier was placed inside the dish to create the wound. The results indicated that surface coating did not affect cell proliferation/viability, and the wound-healing rate increased in coated surfaces compared to uncoated ones. The present study provides a platform for further understanding the mechanisms of substrate coating-dependent wound-healing processes.
PubMed: 31470524
DOI: 10.3390/ma12172775 -
SLAS Technology Apr 2023The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and...
The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and environmental stresses on cell growth and survival. Automation of the spot assay experiments from preparing a dilution series to spotting to observing spots continuously has been implemented based on large laboratory automation devices and robots, especially for high-throughput functional screening assays. However, there has yet to be an affordable solution for the automated spot assays suited to researchers in average laboratories and with high customizability for end-users. To make reproducible spot assay experiments widely available, we have automated the plate-based yeast spot assay of budding yeast using Opentrons OT-2 (OT-2), an affordable liquid-handling robot, and a flatbed scanner. We prepared a 3D-printed mount for the Petri dish to allow for precise placement of the Petri dish inside the OT-2. To account for the uneven height of the agar plates, which were made by human hands, we devised a method to adjust the z-position of the pipette tips based on the weight of each agar plate. During the incubation of the agar plates, a flatbed scanner was used to automatically take images of the agar plates over time, allowing researchers to quantify and compare the cell density within the spots at optimal time points a posteriori. Furthermore, the accuracy of the newly developed automated spot assay was verified by performing spot assays with human experimenters and the OT-2 and quantifying the yeast-grown area of the spots. This study will contribute to the introduction of automated spot assays and the automated acquisition of growth processes in conventional laboratories that are not adapted for high-throughput laboratory automation.
Topics: Humans; Saccharomyces cerevisiae; Agar; Robotics; Automation; Genotype
PubMed: 36503082
DOI: 10.1016/j.slast.2022.12.001 -
Journal of Visualized Experiments : JoVE Feb 2020Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of...
Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated Rac increase differentiation, high Rac levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of Rac.
Topics: Animals; Cadherins; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Culture Media; Epithelial Cells; Female; Mammary Glands, Animal; Mice; Milk Proteins; Spheroids, Cellular; rac GTP-Binding Proteins
PubMed: 32176212
DOI: 10.3791/60147 -
Sensors (Basel, Switzerland) Oct 2023Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and...
Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and segmentation-driven approaches have been proposed by researchers. However, U-Net remains the most frequently cited and used deep learning method in these domains. The latter approach provides a segmentation output map and requires an additional counting procedure to calculate unique segmented regions and detect microbial colonies. However, due to pixel-based targets, it tends to generate irrelevant artifacts or errant pixels, leading to inaccurate and mixed post-processing results. In response to these challenges, this paper proposes a novel hybrid counting approach, incorporating a multi-loss U-Net reformulation and a post-processing Petri dish localization algorithm. Firstly, a unique innovation lies in the multi-loss U-Net reformulation. An additional loss term is introduced in the bottleneck U-Net layer, focusing on the delivery of an auxiliary signal that indicates where to look for distinct CFUs. Secondly, the novel localization algorithm automatically incorporates an agar plate and its bezel into the CFU counting techniques. Finally, the proposition is further enhanced by the integration of a fully automated solution, which comprises a specially designed uniform Petri dish illumination system and a counting web application. The latter application directly receives images from the camera, processes them, and sends the segmentation results to the user. This feature provides an opportunity to correct the CFU counts, offering a feedback loop that contributes to the continued development of the deep learning model. Through extensive experimentation, the authors of this paper have found that all probed multi-loss U-Net architectures incorporated into the proposed hybrid approach consistently outperformed their single-loss counterparts, as well as other comparable models such as self-normalized density maps and YOLOv6, by at least 1% to 3% in mean absolute and symmetric mean absolute percentage errors. Further significant improvements were also reported through the means of the novel localization algorithm. This reaffirms the effectiveness of the proposed hybrid solution in addressing contemporary challenges of precise in vitro CFU counting.
PubMed: 37837169
DOI: 10.3390/s23198337 -
Asian Pacific Journal of Allergy and... Dec 2023Cytokine-induced killer (CIK) cells are ex-vivo expanded T cells which present a phenotype of both T and Natural Killer cell properties.
BACKGROUND
Cytokine-induced killer (CIK) cells are ex-vivo expanded T cells which present a phenotype of both T and Natural Killer cell properties.
OBJECTIVE
To compare the proliferation and functional properties of human CIK cells cultured in three cell culture plasticwares.
METHODS
The number and viability of CIK cells were monitored. The expression of surface markers (CD3 and CD56), TH1 cytokines (IFN-γ and TNF-α), and cytolytic granules (granzyme B and perforin) were determined by flow cytometry.
RESULTS
The number of CIK cells cultured in a static bag was highest compared to those in a petri dish and gas-permeable flask. However, CIK cells cultured in all plasticwares similarity expressed surface marker, TH1 cytokines, and cytolytic granules.
CONCLUSIONS
Considering safety, efficacy, and cost, a static bag is the best plasticware for culturing CIK cells.
Topics: Humans; Cytokine-Induced Killer Cells; Cells, Cultured; Interferon-gamma; Cytokines; Cell Culture Techniques
PubMed: 33274953
DOI: 10.12932/AP-140720-0913 -
ACS Nano Sep 2023Surfaces contaminated with pathogens raise concerns about the increased risk of disease transmission and infection. To clean biocontaminated surfaces, alcohol-based...
Surfaces contaminated with pathogens raise concerns about the increased risk of disease transmission and infection. To clean biocontaminated surfaces, alcohol-based disinfectants have been predominantly used for disinfecting high-touch areas in diverse settings. However, due to its limited antimicrobial activities and concern over the emergence of alcohol-tolerant strains, much effort has been made to develop highly efficient disinfectant formulations. In this study, we hypothesize that the addition of a physical pathogen inactivation mechanism by salt recrystallization (besides the existing chemical inactivation mechanism by alcohol in such formulations) can improve inactivation efficiency by preventing the emergence of alcohol tolerance. To this end, we employed the drying-induced salt recrystallization process to implement the concept of highly efficient alcohol-based disinfectant formulations. To identify the individual and combined effects of isopropyl alcohol (IPA) and NaCl, time-dependent morphological/structural changes of various IPA solutions containing NaCl have been characterized by optical microscopy/X-ray diffraction analysis. Their antimicrobial activities have been tested on surfaces (glass slide, polystyrene Petri dish, and stainless steel) contaminated with Gram-positive/negative bacteria (methicillin-resistant , , and subsp. Typhimurium) and viruses (A/PR8/34 H1N1 influenza virus and HCoV-OC43 human coronavirus). We found that additional salt crystallization during the drying of the alcohol solution facilitated stronger biocidal effects than IPA-only formulations, regardless of the types of solid surfaces and pathogens, including alcohol-tolerant strains adapted from wild-type MG1655. Our findings can be useful in developing highly effective disinfectant formulations by minimizing the use of toxic antimicrobial substances to improve public health and safety.
Topics: Humans; Disinfectants; Methicillin-Resistant Staphylococcus aureus; Sodium Chloride; Influenza A Virus, H1N1 Subtype; Anti-Infective Agents; Ethanol; 2-Propanol; Escherichia coli
PubMed: 37639494
DOI: 10.1021/acsnano.3c03315 -
Scientific Reports Jul 2020Plant growth promoting rhizobacteria (PGPR) are a functionally diverse group of microbes having immense potential as biostimulants and stress alleviators. Their...
Plant growth promoting rhizobacteria (PGPR) are a functionally diverse group of microbes having immense potential as biostimulants and stress alleviators. Their exploitation in agro-ecosystems as an eco-friendly and cost-effective alternative to traditional chemical inputs may positively affect agricultural productivity and environmental sustainability. The present study describes selected rhizobacteria, from a range of origins, having plant growth promoting potential under controlled conditions. A total of 98 isolates (ectophytic or endophytic) from various crop and uncultivated plants were screened, out of which four endophytes (n, L, K and Y) from Phalaris arundinacea, Solanum dulcamara, Scorzoneroides autumnalis, and Glycine max, respectively, were selected in vitro for their vegetative growth stimulating effects on Arabidopsis thaliana Col-0 seedlings with regard to leaf surface area and shoot fresh weight. A 16S rRNA gene sequencing analysis of the strains indicated that these isolates belong to the genera Pseudomonas, Bacillus, Mucilaginibacter and Rhizobium. Strains were then further tested for their effects on abiotic stress alleviation under both Petri-plate and pot conditions. Results from Petri-dish assay indicated strains L, K and Y alleviated salt stress in Arabidopsis seedlings, while strains K and Y conferred increases in fresh weight and leaf area under osmotic stress. Results from subsequent in vivo trials indicated all the isolates, especially strains L, K and Y, distinctly increased A. thaliana growth under both normal and high salinity conditions, as compared to control plants. The activity of antioxidant enzymes (ascorbate peroxidase, catalase and peroxidase), proline content and total antioxidative capacity also differed in the inoculated A. thaliana plants. Furthermore, a study on spatial distribution of the four strains, using either conventional Petri-plate counts or GFP-tagged bacteria, indicated that all four strains were able to colonize the endosphere of A. thaliana root tissue. Thus, the study revealed that the four selected rhizobacteria are good candidates to be explored as plant growth stimulators, which also possess salt stress mitigating property, partially by regulating osmolytes and antioxidant enzymes. Moreover, the study is the first report of Scorzoneroides autumnalis (fall dandelion) and Solanum dulcamara (bittersweet) associated endophytes with PGP effects.
Topics: Arabidopsis; Arabidopsis Proteins; Bacteria; Endophytes; Gene Expression Regulation, Plant; Osmotic Pressure; Phylogeny; Plant Leaves; Plant Shoots; RNA, Ribosomal, 16S; Salt Stress; Soil Microbiology
PubMed: 32728116
DOI: 10.1038/s41598-020-69713-5 -
Angewandte Chemie (International Ed. in... Mar 2023Quantifying glutathione (GSH) in cells and organisms is of great significance for understanding the mechanism of oxidative stress in various physiological and...
Quantifying glutathione (GSH) in cells and organisms is of great significance for understanding the mechanism of oxidative stress in various physiological and pathological processes. However, the quantification by fluorescence bioimaging in living tissues has much stricter requirements than the "Petri dish"-cultured cells in flat plates. Based on the evaluation of the electronic structure and steric hindrance-tuned reactivity of phospha-substituted rhodamine with GSH, a reversible Förster resonance energy transfer (FRET) probe ZpSiP with a distinct performance (K =4.9 mM, t =0.57 s, k=81 M s ) is developed for real time quantifying GSH in living cells. Furthermore, the near-infrared (NIR) probe succeeded in sensitively tracking the dynamics of GSH in the real organisms bearing tumors, chronic renal failure, and liver fibrosis for unveiling the related pathological processes. We believe that the advance in chemistry with quantitative analysis methods will initiate more promising progress and broad applications.
Topics: Fluorescent Dyes; Rhodamines; Fluorescence Resonance Energy Transfer; Glutathione; Limit of Detection
PubMed: 36564368
DOI: 10.1002/anie.202217326 -
Veterinary Parasitology Dec 2021This study was carried out aiming to evaluate the repellent and acaricidal activity of major ingredient compounds from coconut oil including their methyl ester...
This study was carried out aiming to evaluate the repellent and acaricidal activity of major ingredient compounds from coconut oil including their methyl ester derivatives and catnip oil against nymphs and larvae of Amblyomma sculptum. Repellent candidates, coconut oil free fatty acids (coconut FFA mainly C, C and C acid); lauric acid (C acid); capric acid (C acid); methyl laurate; methyl caprate and 10 % each of C, C and C acid (1:1:1) in lavender oil formulation (CFA in lavender formula) and catnip oil (Nepeta cataria), were screened using a Petri dish bioassay to assess repellency. Catnip oil, methyl caprate, methyl laurate, and CFA in lavender formulation repelled ticks strongly (P < 0.05) at almost all times evaluated, with an average of 77.8-100% repellency. Some candidate repellents with consistent strong repellence observed were selected for further evaluation, with coconut CFA in lavender formula showing a repellency lasted up to 7 days, while those of catnip oil and methyl caprate were active for 4 and 3 days, respectively. For the acaricide test, five concentrations (2.5; 5; 10; 15 and 20 mg/mL) were evaluated using the larval packet test. Only CFA in lavender formula and two methyl esters showed acaricidal activity, with methyl laurate presenting the strongest toxicity at 15 mg/mL concentration, which was effective against more than 93 % of the tested larvae. Catnip oil caused no mortality of A. scultptum larvae in all concentrations tested.
Topics: Acaricides; Amblyomma; Animals; Coconut Oil; Insect Repellents; Lauric Acids; Nepeta
PubMed: 34678676
DOI: 10.1016/j.vetpar.2021.109591