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Protein Science : a Publication of the... Apr 2016From humble beginnings of a contaminated petri dish, β-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The... (Review)
Review
From humble beginnings of a contaminated petri dish, β-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an "arms race" with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever-dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in β-lactam research. Here the current state of β-lactam antibiotics from a structural perspective was reviewed.
Topics: Anti-Bacterial Agents; Humans; Structure-Activity Relationship; beta-Lactam Resistance; beta-Lactamase Inhibitors
PubMed: 26813250
DOI: 10.1002/pro.2889 -
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 -
Investigative Ophthalmology & Visual... Nov 2017To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to...
PURPOSE
To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to intraocular pressure (IOP) and renews the medium.
METHODS
A BR comprising endothelial and epithelial chambers was machined in a biocompatible material. The porcine cornea, securely held, separated the chambers. Medium flow and pressure inside the endothelial chamber were maintained by a peristaltic pump. In the epithelial chamber, the corneal surface was alternatively exposed to air and a specific medium. Two transparent windows allowed thickness measurement by optical coherence tomography without opening the BR. Porcine corneas stored in the BR-on (pressure 20 mm Hg, flow 5 μL/min, temperature 31°C) were compared with (1) BR-off (no pressure or flow); (2) organ culture; and (3) Petri dish with agar on the endothelial side. Epithelial and limbal structure and differentiation, corneal transparency and thickness, and endothelial viability were compared after 7 days of storage and with fresh corneas.
RESULTS
Corneas stored in the BR-on were thinner and more transparent than those stored with the other methods. The BR-on preserved a stratified and differentiated (K3/K12+) corneal epithelium and undifferentiated basal limbal cells with stemness markers (K3/K12-; ABCB5, K14, p75+), as well as endothelial integrity.
CONCLUSIONS
By recreating equivalent IOP and medium renewal, the BR obtained unprecedented storage quality of porcine corneas and preserved their main epithelial, limbal, and endothelial characteristics.
Topics: Animals; Bioreactors; Cell Count; Cell Differentiation; Cell Survival; Cornea; Equipment Design; Models, Animal; Organ Culture Techniques; Organ Preservation; Swine
PubMed: 29164231
DOI: 10.1167/iovs.17-22218 -
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 -
Journal of Visualized Experiments : JoVE May 2019Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the...
Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4-5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.
Topics: Animals; Female; Genotyping Techniques; Male; Polymerase Chain Reaction; Sea Anemones
PubMed: 31132068
DOI: 10.3791/59541 -
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 -
Environmental Health Insights 2022Indoor air quality determines the well-being of occupants. It has been linked to sick building syndrome and building-related diseases which lead to many socio-economic...
Indoor air quality determines the well-being of occupants. It has been linked to sick building syndrome and building-related diseases which lead to many socio-economic problems including reduced productivity and impaired learning. Indoor air quality problem is more serious for prisoners, due to their confinement and exposure condition. However, it has not been studied in our study setting. Thus, this study aimed to determine the indoor air microbial quality and associated factors in Jimma town prison administration, Southwestern Ethiopia. A cross-sectional study design was employed in August 2021. Data on the general condition of the prison rooms and occupancy were collected by trained data collectors using an observational checklist. The microbial sample was collected using a sterilized Petri dish. A total of 19 triplicate air samples were collected using Mannitol salt agar and Sabouroad dextrose agar media for the growth of and fungi respectively. Data were analyzed using SPSS version 23 and presented using tables and a graph. The effect of predictor variables on the microbial load was also analyzed by using linear regression. The finding of this study revealed that the microbial load of indoor air at Jimma town prison administration ranged from 891 to 15 439 and 315 to 3067 CFU/m³ for and fungi respectively. Both and the fungal load of the indoor environment were positively affected by the temperature of the room. Whereas, the floor space per inmate affects the concentration of alone. Almost all rooms of the prison administration had microbial load beyond the acceptable limit. Higher temperature, less floor space per inmate, bad floor cleanness conditions, inadequate ventilation, and dampness were contributing factors to the high load of and fungus. Thus, additional rooms are required to reduce overcrowding and keep room temperature.
PubMed: 36003416
DOI: 10.1177/11786302221118842 -
Microbiome Oct 2015Settled airborne dust is used as a surrogate for airborne exposure in studies that explore indoor microbes. In order to determine whether detecting differences in dust...
BACKGROUND
Settled airborne dust is used as a surrogate for airborne exposure in studies that explore indoor microbes. In order to determine whether detecting differences in dust environments would depend on the sampler type, we compared different passive, settled dust sampling approaches with respect to displaying qualitative and quantitative aspects of the bacterial and fungal indoor microbiota.
RESULTS
Settled dust sampling approaches-utilizing plastic petri dishes, TefTex material, and electrostatic dustfall collectors (EDCs)-were evaluated in indoor spaces in the USA and Finland and in an experimental chamber study. The microbial content was analyzed with quantitative PCR (qPCR) to quantify total bacterial and fungal biomass and through high-throughput sequencing to examine bacterial community composition. Bacterial composition and diversity were similar within a sampling environment regardless of the sampler type. The sampling environment was the single largest predictor of microbial community composition within a study, while sampler type was found to have much less predictive power. Quantitative analyses in indoor spaces indicated highest yields using a petri dish approach, followed by sampling with EDCs and TefTex. The highest correlations between duplicate samples were observed for EDC and petri dish approaches, indicating greater experimental repeatability for these sampler types. For the EDC samples, it became apparent that, due to the fibrous nature of the material, a rigorous extraction protocol is crucial to obtain optimal yields and stable, repeatable results.
CONCLUSIONS
Correlations between sampler types were strong both in compositional and quantitative terms, and thus, the particular choice of passive settled dust sampler is not likely to strongly alter the overall conclusion of a study that aims to characterize dust across different environments. Microbial cell abundances determined from settled dust varied with the use of different sampling approaches, and thus, consistency in the method is necessary to allow for absolute comparisons within and among studies. Considering practical aspects, petri dishes were found to be an inexpensive, simple, and feasible approach that showed the highest quantitative determinations under typical building conditions, though the choice of sampler will ultimately depend on study logistics and characteristics such as low- or high-exposure settings.
Topics: Air Microbiology; Air Pollution, Indoor; Bacteria; Biodiversity; Dust; Environmental Monitoring; Finland; Fungi; Humans; Microbiota; United States
PubMed: 26434807
DOI: 10.1186/s40168-015-0112-7