Optimization of Gamma Aminobutyric Acid Production Using High Pressure Processing (HPP) by Lactobacillus brevis PML1

In the present research, the production potential of gamma aminobutyric acid (GABA) using Lactobacillus brevis PML1 was investigated. In addition, the microorganism viability was examined in MAN, ROGOSA, and SHARPE (MRS) after undergoing high hydrostatic pressure at 100, 200, and 300 MPa for 5, 10, and 15 min. Response surface methodology (RSM) was applied to optimize the production conditions of GABA as well as the bacteria viability. Analysis of variance (ANOVA) indicated that both the independent variables (pressure and time) significantly influenced the dependent ones (GABA and bacteria viability) ( P < 0.05 ). The optimum extraction conditions to maximize the production of GABA included the pressure of 300 MPa and the time of 15 min. The amount of the compound was quantified using thin-layer chromatography (TLC) and spectrophotometry. For the process optimization, a central composite design (CCD) was created using Design Expert with 5 replications at the center point, whereby the highest content of GABA was obtained to be 397.73 ppm which was confirmed by high performance liquid chromatography (HPLC). Moreover, scanning electron microscopy (SEM) was utilized to observe the morphological changes in the microorganism. The results revealed that not only did have Lactobacillus brevis PML1 the potential for the production of GABA under conventional conditions (control sample) but also the content of this bioactive compound could be elevated by optimizing the production parameters.

EasyFlow: User-friendly Workflow for Image-based Droplet Analysis with Multipurpose Modules

Droplet-based experimental platforms allow researchers to perform massive parallelization and high-throughput studies, such as single-cell experiments. Even though there are various options of image analysis software to evaluate the experiment, selecting the right tools require experience and is time consuming. Experts and sophisticated workflow are required to perform the analysis, especially to detect the droplets and analyze their content. There is need for user-friendly droplet analysis pipelines that can be adapted in laboratories with minimum learning curve. Here, we provide a user-friendly workflow for image-based droplet analysis. The workflow comprises of a) CellProfiler-based image-analysis pipeline and b) accompanied with web application that simplifies the analysis and visualization of the droplet-based experiment. We construct necessary modules in CellProfiler (CP) to detect droplets and export the results into our web application. Using the web application, we are able to process and provide basic profiles of the droplet experiment (droplet sizes, droplet signals, sizes-signals plot, and strip plot for each label/condition). We also add a specific module for growth heterogeneity studies in bacteria populations that includes single cell viability analysis and probability distribution of minimum inhibition concentration (MIC) values in population. Our pipeline is usable for both poly- and monodisperse droplet emulsions.

Quantitative Action Spectroscopy Reveals ARPE-19 Sensitivity to Long-Wave Ultraviolet Radiation at 350 nm and 380 nm

The role of ultraviolet radiation (UVR) exposure in the pathology of age-related macular degeneration (AMD) has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to AMD-specific parameters of tissue function. We, therefore, explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced AMD, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight. Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10nm bands of UVR between 290 and 405nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR. Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350nm as well as 380nm. Effects at 350nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress. We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350nm and somewhat less at 380nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of AMD.

Establishment and Functional Characterization of Immortalized Rabbit Dermal Papilla Cell Lines

Background Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in the HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. Method In this study, we introduced SV40LT into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DP cell lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). Result These cell lines displayed early passage morphology and displayed high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA ,IGF1, ALPL, FGF2, BMP2 and TGFβ2; α-SMA and VIM protein), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. Conclusion The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

A viability analysis of sustainable by-catch fisheries under direct conservation measures

<p>Sustainability in fisheries is a complex concept and one that has attracted a rich history of research over time. The basic concerns of sustainability are how to reconcile ecological, economic and social requirements within the perspectives of intra- and inter-generational equity. Therefore, maintaining these requirements simultaneously is critical to achieving a perennial system and avoiding so-called “crisis” situations. It is contended that viability theory, which is a relatively new area of mathematics, rigorously captures the essence of sustainability. Using viability theory, this thesis develops two viability models based on different direct conservation measures (i.e. input and output controls) to examine the feasibility conditions under which a regulator can achieve sustainability in a fishery characterised by a “by-catch process”, whereby one species is targeted and another species is incidentally caught as by-catch. The first model considers a by-catch fishery where fishing input is controlled by a regulator. The second model considers two interrelated fisheries managed using a dual quantity-price system, which is based on New Zealand’s Quota Management System (QMS). For each model, the set of constraints representing the “good health” of the system are characterised using managerial priorities identified in the literature. Then, the viability kernel, which is the largest set of initial states for which there are controls that result in inter-temporal trajectories satisfying all the constraints, is approximated numerically. This is achieved by employing VIKAASA, which is a computer application capable of generating kernel approximations. The viability kernel provides the regulator with meaningful reference values and indicators for desirable or undesirable states of the fishery, which serve as important inputs into policy decisions. This study also shows the potential for viability theory to provide policy makers with a better insight of how to integrate ecosystem considerations into the QMS.</p>

A viability analysis of sustainable by-catch fisheries under direct conservation measures

<p>Sustainability in fisheries is a complex concept and one that has attracted a rich history of research over time. The basic concerns of sustainability are how to reconcile ecological, economic and social requirements within the perspectives of intra- and inter-generational equity. Therefore, maintaining these requirements simultaneously is critical to achieving a perennial system and avoiding so-called “crisis” situations. It is contended that viability theory, which is a relatively new area of mathematics, rigorously captures the essence of sustainability. Using viability theory, this thesis develops two viability models based on different direct conservation measures (i.e. input and output controls) to examine the feasibility conditions under which a regulator can achieve sustainability in a fishery characterised by a “by-catch process”, whereby one species is targeted and another species is incidentally caught as by-catch. The first model considers a by-catch fishery where fishing input is controlled by a regulator. The second model considers two interrelated fisheries managed using a dual quantity-price system, which is based on New Zealand’s Quota Management System (QMS). For each model, the set of constraints representing the “good health” of the system are characterised using managerial priorities identified in the literature. Then, the viability kernel, which is the largest set of initial states for which there are controls that result in inter-temporal trajectories satisfying all the constraints, is approximated numerically. This is achieved by employing VIKAASA, which is a computer application capable of generating kernel approximations. The viability kernel provides the regulator with meaningful reference values and indicators for desirable or undesirable states of the fishery, which serve as important inputs into policy decisions. This study also shows the potential for viability theory to provide policy makers with a better insight of how to integrate ecosystem considerations into the QMS.</p>

No Correlation Between Pollen Fertility and Viability: Differential Measures of Male Gametophytic Fitness in Cannabis sativa L.

Pollen grains are male gametophytes, an ephemeral haploid generation of plants, commonly engaging in competition for a limited supply of ovules. Since differential male fertility may influence the direction and pace of population evolution, the relative fitness of pollen is regularly estimated as either pollen viability, the proportion of pollen containing intact cytoplasm's and regenerative nuclei, or pollen fertility, the frequency of pollen germinating under standardized conditions. Here, we estimated the relative fitness of pollen in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing pollen fertility and viability from multiple sires. Pollen fertility quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen viability declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both fertility and viability analysis displayed no significant correlation, implying researchers cannot predict pollen fertility from pollen viability, nor infer male gametophytic fitness from a single measure. Our work demonstrates two approaches to measure proxies of male fitness in C. sativa, and identifies new questions around what are valuable estimates of male fitness in plants.