Influence of Storage Temperature, Moisture Content, and Physical Impurities on the Distribution and Survival of Salmonella enterica in Poultry Fat Intended for Pet Food Use

2018 ◽  
Vol 81 (8) ◽  
pp. 1364-1372 ◽  
Author(s):  
RINARA C. KIEL ◽  
JENNIFER N. MARTIN ◽  
DALE R. WOERNER ◽  
RACHEL MURPHY ◽  
IFIGENIA GEORNARAS ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Moisture Content ◽  
Fluorescent Protein ◽  
Storage Temperature ◽  
Impurity Level ◽  
Compositional Variation ◽  
Pet Food ◽  
Poultry Fat ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Contamination of rendered products with Salmonella is a concern for the rendering industry, particularly when those products are intended for use in other foodstuffs, such as pet food. This study was conducted to understand the influence of compositional variation on the location and survivability of Salmonella in a poultry fat matrix. Specifically, this study aimed to (i) assess the influence of postinoculation time and moisture content on the distribution of Salmonella in rendered poultry fat and (ii) evaluate the impact of postinoculation time and physical parameters (i.e., impurity level and moisture content) on survival of three Salmonella strains in rendered poultry fat stored at two different temperatures. Three studies, designated as study I(a), I(b), and II, respectively, were conducted to address these objectives. In study I(a), a green fluorescent protein–expressing strain of Salmonella Typhimurium was used to map the organism within warmed (45°C) poultry fat containing various levels of moisture. In study I(b), the influence of storage temperature on the survivability of green fluorescent protein–expressing Salmonella was evaluated. In study II, the impacts of physical impurities, moisture content, and storage temperature on the survivability of three Salmonella strains (Enteritidis, Senftenberg, and Typhimurium) were assessed. The results of this study demonstrated that composition (i.e., moisture and impurity contents) influences the survivability of Salmonella in poultry fat; specifically, Salmonella is more persistent in poultry fat with a greater moisture content and water activity. Nonetheless, although composition impacts the distribution and survivability of Salmonella in poultry fat, Salmonella generally does not survive in poultry fat maintained at high temperatures (45°C and above).

scholarly journals Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments

2010 ◽  
Vol 76 (18) ◽  
pp. 6119-6127 ◽  
Author(s):  
Putthapoom Lumjiaktase ◽  
Claudio Aguilar ◽  
Tom Battin ◽  
Kathrin Riedel ◽  
Leo Eberl
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Natural Habitat ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Broad Host Range ◽  
Construction Of Self ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

scholarly journals Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

2012 ◽  
Vol 140 (3) ◽  
pp. 249-266 ◽  
Author(s):  
Mehdi Najafi ◽  
Mohammad Haeri ◽  
Barry E. Knox ◽  
William E. Schiesser ◽  
Peter D. Calvert
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Diffusion ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Live Cell ◽  
Confocal Imaging ◽  
Slow Phase ◽  
Protein Diffusion ◽  
Green Fluorescent ◽  
The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

scholarly journals Survival of Salmonella Transformed To Express Green Fluorescent Protein on Italian Parsley as Affected by Processing and Storage

2005 ◽  
Vol 68 (4) ◽  
pp. 687-695 ◽  
Author(s):  
E. A. DUFFY ◽  
L. CISNEROS-ZEVALLOS ◽  
A. CASTILLO ◽  
S. D. PILLAI ◽  
S. C. RICKE ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Storage Temperature ◽  
Control Point ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser ◽  
Processing And Storage ◽  
Green Fluorescent ◽  
And Storage

To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35°C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25°C and over 0, 1, 7, 14, and 30 days of storage at 4°C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25°C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4°C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants

2020 ◽  
Vol 33 ◽  
Author(s):  
Majid Masso
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Classification Performance ◽  
Machine Learning Algorithms ◽  
Sequence Structure ◽  
Aequorea Victoria ◽  
Function Relationship ◽  
Green Fluorescent ◽  
The Impact ◽  
Computational Mutagenesis

Abstract A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure–function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson’s correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.

Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein

2010 ◽  
Vol 299 (6) ◽  
pp. C1318-C1323 ◽  
Author(s):  
Eiji Takahashi ◽  
Michihiko Sato
Keyword(s):  
Green Fluorescent Protein ◽  
Diffusion Model ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Red Shift ◽  
Two Dimensional ◽  
Fluorescence Measurements ◽  
Hep3b Cells ◽  
Green Fluorescent ◽  
The Impact

Gradients of Po2 between capillary blood and mitochondria are the driving force for diffusional O2 delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O2 gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O2 gradients, we developed a novel technique that permits imaging of intracellular O2 levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O2 ≤3%, by the O2-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O2 diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O2 diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O2, respectively. Thus the average O2 gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O2 gradients is intracellular. Findings in the present two-dimensional O2 diffusion model support the crucial role of tissue O2 diffusion in defining the O2 microenvironment in individual cells.

scholarly journals Estimating numbers of intracellular molecules through analysing fluctuations in photobleaching

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Wide Field ◽  
Order Of Magnitude ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact ◽  
Quantitative Fluorescence

Abstract The impact of fluorescence microscopy has been limited by the difficulties of expressing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we propose an estimator to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. Performing experiments in budding yeast, we show that our estimates of numbers agree, within an order of magnitude, with published biochemical measurements, for all six proteins tested. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

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