Physiological State Of Cells Research Articles

Growth-dependent photoinactivation kinetics of Enterococcus faecalis.

To investigate how the growth stage of Enterococcus faecalis affects its photoinactivation in clear water. Enterococcus faecalis were grown in batch cultures to four different growth stages or grown in chemostats set at four different dilution rates, then harvested and exposed to full spectrum or UVB-blocked simulated sunlight. Experiments were conducted in triplicate in clear water with no added sensitizers. Decay curves were shoulder-log linear and were generally not statistically different in experiments conducted under full spectrum light. Shoulders were longer and first order inactivation rates smaller when experiments were seeded with cells grown to stationary as compared to exponential phase, and for slower growing cells when experiments were done under UVB-blocked light. Chemostat-sourced bacteria generally showed less variability among replicates than batch-sourced cells. The physiological state of cells and the method via which they are being generated may affect the photoinactivation experimental results. Photoinactivation experiments conducted with exponential phase cells may overestimate the photoinactivation kinetics in the environment, particular if UVB-independent mechanisms predominate. Chemostat-sourced cells are likely to provide more consistent experimental results than batch-sourced cells.

Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

Hypoxia in breast cancer.

Breast cancer continues to be one of the most common malignancies and is a serious problem particularly in women, although men may also be affected. Such lesions are commonly accompanied by hypoxia, and therefore hypoxia-dependent mechanisms, such as overexpression of hypoxia-inducible factor (HIF), the mechanisms are studied as part of the search for a novel method of cancer treatment. Blocking the activity of HIF and HIF-dependent molecular changes raises hopes for identification of a molecular target to inhibit the tumor growth or even to completely prevent its progression. However, this is difficult due to the crucial role HIF plays in numerous processes occurring not only in cancer cells but mostly in healthy systemic cells in physiological conditions.

Single cell biotechnology to shed a light on biological 'dark matter' in nature.

It is estimated that over 99% of microorganisms have not as yet been cultivated (Whitman et al., 1998). These uncultured microbes are the ‘dark matter’ of the microbiological world and play important roles in natural ecosystems and the human microbiome (Lagier et al., 2012; Rinke et al., 2013); however, their ecological role and function remain largely elusive (Lasken, 2012; Li et al., 2012a,b). Furthermore, these uncultured bacteria represent a significant, yet largely untapped, genetic resource for use in synthetic biology for the provision of novel bioparts or biobricks, in medicine for new drug biosynthesis, in industry for robust biocatalysts and biofuel synthesis, and in environmental bioremediation for new biodegradation genes. Metagenomics circumvents the cultivation issue by extracting the total DNA from an environmental sample, and directly sequencing it (Handelsman, 2004), and this approach has revealed an unprecedented view of the diversity and complexity of microbial communities. However, such approaches are usually unable to define or validate the specific role of individual members of the usually complex microbiota. Single cell biotechnology, which characterizes microbial cells in their native microbiota one by one, offers a new approach to study uncultured bacteria. An ideal platform is to integrate accurate and ‘contamination-free’ single cell sorting tools with powerful next-generation DNA sequencing. This will usher in a new area of single cell omics (genomics, transcriptomics, proteomics and metabolomics). For single cell biotechnology, there are a number of key challenges: non-invasive and in-vivo cell analysis, the linking of cell phenotypes to specific ecological functions (e.g. substrate metabolism), overcoming limitations in measurement parameters, systematic differentiation of the given ‘state’ or phenotype of a cell and isolation of live single cells from complex samples in-situ. Among the various single cell sorting techniques (Lasken, 2012; Li et al., 2012a,b), an emerging approach is Raman-activated cell sorting (RACS), which overcomes the requirement for external labelling. Single cell Raman spectra (SCRS) provide a label-free, non-invasive and intrinsic phenotypic profile of individual cells which can be used to characterize cell type, physiological state and cell functionalities (Huang et al., 2004; 2007a,b,c; Harz et al., 2009; Li et al., 2012a,b; Wang et al., 2014). A typical SCRS provides an intrinsic chemical ‘fingerprint’ of a single cell, and usually contains multi-parameter (> 1000 readings) including rich information on nucleic acids, protein, carbohydrates and lipids (Li et al., 2012a,b). Since SCRS measures the vibration of molecular bonds, it is sensitive to stable isotope compounds and SCRS undergoes Raman shift when cells incorporate stable isotope compounds (e.g. 13C-, 15N-substrates or 2H from heavy water D2O) into the cell's building blocks (e.g. DNA, lipids, protein or carbohydrate) (Huang et al., 2004; 2007a,b,c; Wang et al., 2013). SCRS offers a unique way to link cells to specific functions (e.g. C/N metabolism and metabolic activity) and to define cells of interest at a single cell level. A RACS system consists of a SCRS detection system and a cell isolation system that can be optical tweezers (Huang et al., 2009), a microfluidic device (Li et al., 2012a,b) or a single cell ejection system (Wang et al., 2013). RACS would identify cells of interest and isolate them for downstream single cell omics analysis. The isolated single cells would be processed on microfluidic chips for DNA/RNA extraction and amplification. The DNA/RNA can then be quantified or sequenced to decode the genomes or transcriptomes of the particular cells. Such a platform directly establishes the links between genotype and phenotype of individual cells, thus offering unprecedented opportunities to study how variability of environmental and genetic impacts on the phenotype of single cells. Single cell biotechnology will not only be a powerful tool to microbiology, but also herald single cell biology as a new frontier of cell biology. A single cell is the basic functional unit of life and all living organisms start from single cells. Learning how cells work by studying the individual cell is an important component of cell biology and single cell technology promotes a deeper understanding of cell biology. Recent research from the studies of single cells reveals that individual cells within the same population may differ dramatically in function, and these differences have profound biological implications, ranging from bacterial physiology to embryotic cell development, tissue differentiation, cancer cell formation and evolution. In summary, during the next decade, just like DNA sequencing, single cell biotechnologies are expected to rapidly move into bench tops of biology laboratory, and to permeate all branches of life sciences and biotechnology. They promise to uncover fundamental biological principles and ultimately improve the diagnosis and treatment of diseases, unravel the ecological role of bacteria in soils, plants and humans, and promote the discovery of new gene functions for use in industry.

Interpretation and Integration of 13C-Fluxomics Data

The fluxome, based on 13C flux, is a newly developed important aspect of cellular science, and it is both the final read-out of the physiological state of cells and an extension of both the transcriptome and proteome. This article records the important methods in the technical development of 13C-fluxomics, reviews the significance and progress in the interpretation and integration of fluxomics data, and then explores several directions of vital importance for the interpretation and integration of 13C-fluxomics.

Resveratrol production in bioreactor: Assessment of cell physiological states and plasmid segregational stability

Resveratrol is a plant secondary metabolite commonly found in peanuts and grapevines with significant health benefits. Recombinant organisms can produce large amounts of resveratrol and, in this work, Escherichia coli BW27784 was used to produce resveratrol in bioreactors while monitoring cell physiology and plasmid stability through flow cytometry and real-time qPCR, respectively. Initially, the influence of culture conditions and precursor addition was evaluated in screening assays and the data gathered was used to perform the bioreactor assays, allowing the production of 160μg/mL of resveratrol. Cellular physiology and plasmid instability affected the final resveratrol production, with lower viability and plasmid copy numbers associated with lower yields. In sum, this study describes new tools to monitor the bioprocess, evaluating the effect of culture conditions, and its correlation with cell physiology and plasmid segregational stability, in order to define a viable and scalable bioprocess to fulfill the need for larger quantities of resveratrol.

Phage adsorption on Enteropathogenic and Shiga Toxin-Producing Escherichia coli strains: Influence of physicochemical and physiological factors

Bacteriophages have proved to be useful tools against pathogenic Escherichia coli strains. The first step in the phage life cycle is the adsorption to the host cell surface. In the present work, three Myoviridae phages (DT1, DT5 and DT6) were used to characterize the adsorption process on three pathogenic E. coli strains, namely two Shiga-toxin producing E. coli (STEC) and one enteropathogenic E. coli (EPEC), in several conditions found on food. The influence of Na+, Mg2+, temperature, pH, periodate, proteinase K and physiological cell state on phage adsorption was investigated. The three phages evaluated showed high adsorption rates at pH7.5 and 5.7 while they were moderate at the lowest pH evaluated (4.5). Sodium or magnesium ions were not indispensable for the adsorption of the three phages evaluated. Specifically, phage particles were adsorbed either in the presence or absence of Mg2+, while increasing Na+ concentration resulted in lower adsorption values for all the systems evaluated. Regarding temperature, phage adsorption was slightly affected at 4°C and 50°C, while it reached its maximum at 37°C. Adsorption rates decreased after the thermal inactivation of cells, though, when chloramphenicol (as protein-synthesis inhibitor) was used, adsorption values on treated and untreated cells were similar. In addition, periodate was able to decrease phage adsorption, thus suggesting the receptors were carbohydrates in nature. All these results showed that the adsorption process was only partially affected and most conditions are suitable for the completion of the first step in the phage life cycle. Therefore, phages evaluated in this study can be used to prevent foodborne diseases on several food matrices since they are active in a wide range of environmental conditions.

Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions

A series of laboratory experiments were conducted to examine effects of biochemical processes on phytoplankton lipids and their stable carbon isotopic compositions. Thalassiosira weissflogii and Emiliania huxleyi were axenically cultivated in batch cultures through exponential growth (phase I) and stationary period (phase II), followed by dark respiration (phase III) and subsequent microbial degradation (phase IV). The results indicated that the bulk parameters, lipid contents and associated isotopic compositions of two phytoplankton species varied differently during different cell growth stages, cell respiration and microbial degradation processes. T. weissflogii continuously produced neutral lipids and fatty acids at similar rates between phase I and phase II. By contrast, E. huxleyi produced neutral lipids at faster rates in phase II than in phase I although the production rates of fatty acids were still similar between the two phases. The lipid isotopic compositions of these two phytoplankton species varied in a similar pattern through cell growth: remarkably positive shift in phase I but relatively small negative shift or little change in phase II. During cell respiration (phase III), two phytoplanktons lost their lipids by varying percentages but the isotopic compositions of all compounds remained relatively constant. With degradation of lipids in phase IV, their isotopic compositions varied in three different ways: positive shift for most fatty acids; little change for sterols and alkenones; and a negative shift for 20:5 fatty acid of T. weissflogii. This experimental study implies: (1) isotopic compositions of phytoplankton lipids are heterogeneously generated through different cell growth phases, likely dependent on cell physiological states, medium conditions, and relative proportions of intracellular components; and (2) microbial degradation plays a more important role in altering isotopic compositions of lipid compounds than cell respiration, probably related to different labilities of lipids between different cellular components.

Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: identifying ChIP-quality p300 monoclonal antibodies.

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1.

Novel magnesium alloy Mg–2La caused no cytotoxic effects on cells in physiological conditions

Using several different in vitro assays, a new biodegradable magnesium alloy Mg–2La, composed of 98% magnesium and 2% lanthanum, was investigated as a possible implant material for biomedical applications. An in vitro cytotoxicity test, according to EN ISO 10993-5/12, with L929 and human osteoblastic cells identified no toxic effects on cell viability at physiological concentrations (at 50% dilutions and higher). The metabolic activity of human osteoblasts in the 100% extract was decreased to <70% and was therefore rated as cytotoxic. The degradation rates of Mg–2La were evaluated in phosphate buffered saline and four different cell culture media. The degradation rates were shown to be influenced by the composition of the solution, and the addition of fetal bovine serum slightly accelerated the corrosive process. The results of these in vitro experiments suggest that Mg–2La is a promising candidate for use as an orthopedic implant material.

Functional significance of macrophage-derived exosomes in inflammation and pain

Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome-mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide-stimulated cells were sufficient to cause nuclear factor-κB activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

Growing Scenedesmus quadricauda in used culture media: is it viable?

Large-scale cultures of freshwater microalgae species demand high volumes of water used for culture media preparation. The recycling of culture media may help decrease cultivation costs, use valuable nutrients that may still remain in the medium after use and make it a green production system. This work investigated the effects of using culture media up to three times to grow Scenedesmus quadricauda. After a first use, culture media were either supplemented with nitrogen (N) and phosphorus (P) or with all nutrients of the medium composition. The effects of used culture media on the algae were verified through chlorophyll a concentrations, number of cells per coenobium, population density, growth rates, and biochemical composition (lipid classes, proteins, and carbohydrates). Cells were harvested through centrifugation before each culture medium use. The results showed that all nutrients should be added to the used medium at each new inoculation; protein/carbohydrate ratios were the best indicator of cell physiological status (higher in controls and lower in used media), and hydrocarbons accounted for 20 % of total lipids in controls and were produced under healthy conditions in S. quadricauda. According to the present results, at least three culture medium uses may be performed without affecting biomass yield, and it may be as promising as fresh medium for general biomass applications, such as biofuels production, but we do not recommend its reuse for fine biomass applications. Even though cells grew well in recycled media with all nutrients added, they were not as healthy as the ones in the control.

Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration

(+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2′-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.

The effect of FISH and CARD-FISH on the isotopic composition of 13C- and 15N-labeled Pseudomonas putida cells measured by nanoSIMS

The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on 13C- and 15N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that 13C and 15N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the 13C and 15N fractions was measured from 94±1.2% and 89.5±3.8% to 90.2±0.8% and 64±4.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the 13C and 15N fractions to 80.7±6.5 and 59.5±4.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.4±3.0% and 47.1±4.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations.

Zmiany w komórkach mezofilu liści roślin Lycopersicon esculentum Mill. spowodowane zasoleniem

Five-week-old tomato plants (Lycopersicon esculentum) cv. Perkoz grown in pots containing garden soil in a growth chamber were submitted to 50 or 150 mM NaCl for 1 h, 2 and 5 days. Tomato leaf anatomy generally did not change after short time salinity, except 5-day-treatment with 150 mM NaCl, where changed cell shape (shrunk and deformed) simultaneously with increased volume of intercellular spaces (IS) were observed. Although leaf hydration (H) depleted only 1 h after 150 mM NaCl treatment both salt concentrations generated two coexisting populations of salt-affected mesophyll cells: (i) slightly-affected (Sl-A) which showed incipient plasmolysis or slightly changed shapes, and (ii) severely-affected (Sv-A) which showed severe plasmolysis; serious deformation of cell shape or disorganization including cell degeneration. In Sl-A cells salinity changed location and shape of chloroplasts which were: more rounded, with oversized starch grains (SG) (2d) or more flat (5d). Salt-mediated changes were becoming more distinguished and pronounced with length of 150 mM NaCl treatment. The amount of salt-affected cells was changing during the experiment and depended on the salt concentration. In 50 mM-treated plants salt-affected cells appeared 1 h after treatment (~40%) and raised up to 78% on 2nd day, however the population of Sl-A cells dominated. In 150 mM NaCl-treated plants the percentage of affected cells raised during the experiment from 75% to 99%. Firstly Sl-A cells dominated, but on the 5th day the majority was Sv-A. Salt-affected cells were distributed quite evenly in palisade or spongy mesophyll, except 2 d after treatment with 50 mM NaCl, when their number was higher in the palisade mesophyll. Sv-A cells in the spongy mesophyll were located mostly near the bundle while in the palisade mesophyll more irregularly. Different susceptibility of cells to salt stress might be the consequence of an unequal distribution of osmotic stress and subsequent ionic stress or physiological state of cells.

Integrative genomics to dissect retinoid functions.

Retinoids and rexinoids, as all other ligands of the nuclear receptor (NR) family, act as ligand-regulated trans-acting transcription factors that bind to cis-acting DNA regulatory elements in the promoter regions of target genes (for reviews see [12, 22, 23, 26, 36]). Ligand binding modulates the communication functions of the receptor with the intracellular environment, which essentially entails receptor-protein and receptor-DNA or receptor-chromatin interactions. In this communication network, the receptor simultaneously serves as both intracellular sensor and regulator of cell/organ functions. Receptors are "intelligent" mediators of the information encoded in the chemical structure of a nuclear receptor ligand, as they interpret this information in the context of cellular identity and cell-physiological status and convert it into a dynamic chain of receptor-protein and receptor-DNA interactions. To process input and output information, they are composed of a modular structure with several domains that have evolved to exert particular molecular recognition functions. As detailed in other chapters in this volume, the main functional domains are the DNA-binding (DBD) and ligand-binding (LBD) [5-7, 38, 56, 71]. The LBD serves as a dual input-output information processor. Inputs, such as ligand binding or receptor phosphorylations, induce allosteric changes in receptor surfaces that serve as docking sites for outputs, such as subunits of transcription and epigenetic machineries or enzyme complexes. The complexity of input and output signals and their interdependencies is far from being understood.

Measurement of phenazines in bacterial cultures.

Certain pseudomonads are capable of producing phenazines-pigmented, reversibly redox-active metabolites that induce a variety of physiological effects on the producing organism as well as others in their vicinity. Environmental conditions and the specific physiological state of cells can dramatically affect the absolute amounts and relative proportions of the various phenazines produced. The method detailed here-high-performance liquid chromatography coupled to detection by UV-Vis absorption-can be used to separate and quantify the amount of phenazines in a Pseudomonas culture. Simple spectrophotometric measurements of filtered culture supernatants can be used to quantify certain oxidized phenazines, such as pyocyanin, in cultures. For cases where the conditions under study are not planktonic cultures (e.g., soil or biofilms) extracting the phenazines may be a necessary first step.

An improved quantitative analysis method for plant cortical microtubules.

The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

Evolution of microbiological analytical methods for dairy industry needs

Traditionally, culture-based methods have been used to enumerate microbial populations in dairy products. Recent developments in molecular methods now enable faster and more sensitive analyses than classical microbiology procedures. These molecular tools allow a detailed characterization of cell physiological states and bacterial fitness and thus, offer new perspectives to integration of microbial physiology monitoring to improve industrial processes. This review summarizes the methods described to enumerate and characterize physiological states of technological microbiota in dairy products, and discusses the current deficiencies in relation to the industry’s needs. Recent studies show that Polymerase chain reaction-based methods can successfully be applied to quantify fermenting microbes and probiotics in dairy products. Flow cytometry and omics technologies also show interesting analytical potentialities. However, they still suffer from a lack of validation and standardization for quality control analyses, as reflected by the absence of performance studies and official international standards.

Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17

Lactobacillus plantarum is a lactic acid bacterium involved in the production of many fermented foods. Recently, several studies have demonstrated that aerobic or respiratory metabolism in this species leads to improved technological and stress response properties. We investigated respiratory growth, metabolite production and stress resistance of Lact.plantarum C17 during batch, fed-batch and chemostat cultivations under respiratory conditions. Sixty mutants were selected for their ability to tolerate oxidative stress using H2 O2 and menadione as selective agents and further screened for their capability to growth under anaerobic, respiratory and oxidative stress conditions. Dilution rate clearly affected the physiological state of cells and, generally, slow-growing cultures had improved survival to stresses, catalase production and oxygen uptake. Most mutants were more competitive in terms of biomass production and ROS degradation compared with wild-type strain (wt) C17 and two of these (C17-m19 and C17-m58) were selected for further experiments. This work confirms that, in Lact.plantarum, respiration and low growth rates confer physiological and metabolic advantages compared with anaerobic cultivation. Our strategy of natural selection successfully provides a rapid and inexpensive screening for a large number of strains and represents a food-grade approach of practical relevance in the production of starter and probiotic cultures.