Sublethal Stress Conditions Research Articles

Mitochondrial ROS driven by NOX4 upregulation promotes hepatocellular carcinoma cell survival after incomplete radiofrequency ablation by inducing of mitophagy via Nrf2/PINK1

BackgroundThe recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear.MethodsHepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives.ResultsHere,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. n-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress.ConclusionThese data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.

Survival of potential probiotic isolated from fermented tea leaf and encapsulated in multilayer beads stored in makiang (Cleistocalyx nervosum var. paniala) juice

Cleistocalyx nervosum var. paniala juice or makiang juice has high content of acid and phenolic compounds which are detrimental to probiotic bacteria. The screening of potential probiotic with acid production and phenolic tolerant bacteria from fermented tea leaves revealed 5 isolates which were identified using 16s rRNA sequencing procedure and found similarity to the strain of Lactobacillus plantarum in GenBank database at 98–100%. The storage of 5 isolates and 6 probiotics in makiang juice for 24 h was indicated that L. plantarum MF105 and TISTR 2074 showed the least reduction of 30.10 ± 1.25 and 61.41 ± 2.37%, respectively. These cultures were encapsulated in 2% alginate bead (Al), Al + single-layer of gelatin (ACG), and Al + double-layer of gelatin (ACGD). They were kept in makiang juice at 4 °C. The physico-chemical characteristics of beads in makiang juice illustrated that the accumulation of phenolic compounds was faster than the pH decreasing and caused initial reduction of bacterial viability. The ACGD beads absorbed slower and accumulated less amount of phenolic compounds than others. They were the most effective in cell protection, the viability of encapsulated MF105 and TISTR2074 were prolonged to more than 7 and 4 days, respectively. Cell adaptation in combination with encapsulation enhanced the survival of probiotic cell in sub-lethal stress conditions. Data had shown that increasing the coating layer provided a chance to improve lethality of probiotic bacteria.

Chronic stress induces formation of stress granules and pathological TDP-43 aggregates in human ALS fibroblasts and iPSC-motoneurons

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43) protein. The RNA-binding protein TDP-43 participates also to cell stress response by forming stress granules (SG) in the cytoplasm to temporarily arrest translation. The hypothesis that TDP-43 pathology directly arises from SG has been proposed but is still under debate because only sub-lethal stress conditions have been tested experimentally so far. In this study we reproduced a mild and chronic oxidative stress by sodium arsenite to better mimic the persistent and subtle alterations occurring during the neurodegenerative process in primary fibroblasts and induced pluripotent stem cell-derived motoneurons (iPSC-MN) from ALS patients carrying mutations in TARDBP and C9ORF72 genes. We found that not only the acute sub-lethal stress usually used in literature, but also the chronic oxidative insult was able to induce SG formation in both primary fibroblasts and iPSC-MN. We also observed the recruitment of TDP-43 into SG only upon chronic stress in association to the formation of distinct cytoplasmic P-TDP-43 aggregates and a significant increase of the autophagy marker p62. A quantitative analysis revealed differences in both the number of cells forming SG in mutant ALS and healthy control fibroblasts, suggesting a specific genetic contribution to cell stress response, and in SG size, suggesting a different composition of these cytoplasmic foci in the two stress conditions. Upon removal of arsenite, the recovery from chronic stress was complete for SG and P-TDP-43 aggregates at 72 h with the exception of p62, which was reduced but still persistent, supporting the hypothesis that autophagy impairment may drive pathological TDP-43 aggregates formation. The gene-specific differences observed in fibroblasts in response to oxidative stress were not present in iPSC-MN, which showed a similar formation of SG and P-TDP-43 aggregates regardless their genotype. Our results show that SG and P-TDP-43 aggregates may be recapitulated in patient-derived neuronal and non-neuronal cells exposed to prolonged oxidative stress, which may be therefore exploited to study TDP-43 pathology and to develop individualized therapeutic strategies for ALS/FTD.

The influence of adaptive stresses on the survival of spray-dried Lactococcus lactis cells.

The research described in this technical research communication examines the hypothesis that sublethal stress conditions can improve the survival of Lactococcus lactis subsp. lactis during drying and subsequent storage. After drying, the L. lactis that had adapted to acid or osmotic stresses did not differ statistically in terms of cell viability loss compared to the control samples tested (~0.38 log cycles). However, the cells that had adapted to oxidative conditions demonstrated a cell viability loss of only 0.01 log cycles. After 45 d of storage at temperatures of 4 and 25 °C, the final L. lactis sample populations were shown to be higher (112.5%) when they had been submitted to sublethal conditions of oxidative stress. When the cell samples were exposed to acid stress conditions, they exhibited a viability loss (0.82 log cycles) that was statistically different from the control sample (0.58 log cycles) after 45 d. Osmotic stress conditions did not demonstrate any influence over cell survival rates. Thus, submitting cells to oxidative stress conditions prior to storage has been shown to be a potential strategy for producing dehydrated cultures of L. lactis strains that are less sensitive to oxygen exposure.

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants.

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious infections and multiple food-borne outbreaks. In this study, we investigated the influence of atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of sigB, rsbR, prfA, gadD, and lmo0799 genes at different treatment time intervals. Further, to ascertain if sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants were similarly affected after 1 min exposure to ACP (p > 0.05), with a difference in response noted only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of treatment (p < 0.05). The results indicate sigB and prfA are important for general stress resistance and biofilm, respectively, loss of these two genes significantly reduced bacterial resistance to ACP treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to cold plasma stress and offers promising insights for optimizing ACP applications.

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested.

Phosphatidylinositol 3-kinase function at very early symbiont perception: a local nodulation control under stress conditions?

Root hair curling is an early and essential morphological change required for the success of the symbiotic interaction between legumes and rhizobia. At this stage rhizobia grow as an infection thread within root hairs and are internalized into the plant cells by endocytosis, where the PI3K enzyme plays important roles. Previous observations show that stress conditions affect early stages of the symbiotic interaction, from 2 to 30 min post-inoculation, which we term as very early host responses, and affect symbiosis establishment. Herein, we demonstrated the relevance of the very early host responses for the symbiotic interaction. PI3K and the NADPH oxidase complex are found to have key roles in the microsymbiont recognition response, modulating the apoplastic and intracellular/endosomal ROS induction in root hairs. Interestingly, compared with soybean mutant plants that do not perceive the symbiont, we demonstrated that the very early symbiont perception under sublethal saline stress conditions induced root hair death. Together, these results highlight not only the importance of the very early host-responses on later stages of the symbiont interaction, but also suggest that they act as a mechanism for local control of nodulation capacity, prior to the abortion of the infection thread, preventing the allocation of resources/energy for nodule formation under unfavorable environmental conditions.

Formação de biofilme por Vibrio parahaemolyticus isolados de pescados

RESUMO: O pescado é um alimento altamente perecível, possui pH próximo a neutralidade, elevada atividade de água e alto teor de nutrientes facilmente utilizáveis por micro-organismos. Vibrio parahaemolyticus pode ser encontrado em ambientes com salinidade entre 3% e 8% e tem pH ideal para multiplicação entre 7,8 e 8,6. É um patógeno que pode causar gastrenterite aguda pelo consumo de frutos do mar contaminados, crus ou mal cozidos. Mesmo os processos de tratamento de água como cloração, adição de antibióticos e filtros apresentam dificuldade em reduzir a contaminação por Vibrio, sendo suposto que este gênero bacteriano pode formar biofilmes em diferentes superfícies. O objetivo do trabalho foi verificar a capacidade de V. parahaemolyticus isolados de pescados formarem biofilme após estresse subletal. No decorrer de um ano, foram realizadas 12 coletas mensais de amostras de peixes capturados no estuário da Lagoa dos Patos, as quais foram analisadas quanto à presença de V. parahaemolyticus. Concomitantemente, foram coletadas assepticamente amostras de água do estuário para análise de sanilidade e pH. Os isolados de Vibrio foram analisados pela reação em cadeia da polimerase (PCR) para identificação da espécie pela presença dos genes toxR. Além dos isolados obtidos no presente trabalho, também foram estudadas 15 outras cepas de V. parahaemolyticus previamente isoladas em outros trabalhos. As cepas foram avaliadas quanto à capacidade de produção de biofilme em placas de microtitulação. A capacidade de produção de biofilme após as cepas serem submetidas a diferentes tipos de estresse subletal (42ºC, 20ºC, 4ºC e pH ácido) também foi testada. Dentre os 120 peixes analisados, foram isolados V. parahaemolyticus de quatro (3,33%) pescados, sendo Mugil platanus a única espécie de peixe na qual o micro-organismo foi encontrado. Das 19 cepas analisadas, 89,5% foram capazes de formar biofilme, o que parece indicar que essa capacidade tem um papel importante na sobrevivência do micro-organismo nos pescados. Dessas, 25% das cepas aumentaram a capacidade de formar biofilme. Com base nos resultados, conclui-se que peixes da espécie M. platanus do estuário da Lagoa dos Patos são hospedeiros de V. parahaemolyticus e que a quase totalidade das cepas são formadoras de biofilme. A exposição a condições subletais de estresse tem efeito distinto sobre as diferentes cepas, induzindo aumento na capacidade de formar biofilme em algumas. Este foi o primeiro estudo realizado com V. parahaemolyticus, para avaliar o efeito de fatores de estresse sobre a formação de biofilme.

Influence of Lactobacillus plantarum WCFS1 on post-acidification, metabolite formation and survival of starter bacteria in set-yoghurt

The objectives of this study were to evaluate the growth and survival of the model probiotic strain Lactobacillus plantarum WCFS1 in co-culture with traditional yoghurt starters and to investigate the impact of preculturing on their survival and metabolite formation in set-yoghurt. L. plantarum WCFS1 was precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor before inoculation in milk. Adaptive responses of L. plantarum WCFS1 were evaluated by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated that sublethal preculturing did not significantly affect survival of L. plantarum WCFS1. On the other hand, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of Lactobacillus delbrueckii subsp. bulgaricus which consequently reduced the post-acidification of yoghurt during refrigerated storage. A complementary metabolomics approach using headspace SPME-GC/MS and 1H NMR combined with multivariate statistical analysis revealed substantial impact of sublethally precultured L. plantarum WCFS1 on the metabolite profiles of set-yoghurt. This study provides insight in the technological implications of non-dairy model probiotic strain L. plantarum WCFS1, such as its good stability in fermented milk and the inhibitory effect on post-acidification.

Proteromorphosis of Neospathodus (Conodonta) during the Permian–Triassic crisis and recovery

The Permian–Triassic evolution of platform conodonts (Gondolellidae) consists mainly of the development of the carina and the platform. During the sublethal environmental stress conditions subsequent to the Permian–Triassic extinction, the Wuchiapingian–Griesbachian Clarkina lineage is replaced by the primitive looking platform-lacking Dienerian–Aegean Neospathodus kummeli–Kashmirella timorensis lineage. It is assumed that, above Jinogondolella denticulata, end of the Capitanian Jinogondolella lineage, “Neospathodus” arcucristatus, an atavistic blade-like homeomorph that lacks a platform, underlies Protoclarkina crofti, of the base of the anagenetic Clarkina lineage. These primitive-looking forms are derived from their immediate ancestors by retrograde evolution, a phenomenon that has been described as proteromorphosis. Such events suggest that proteromorphosis occurs during periods of crisis, with the sudden reappearance of homeomorphic forms that are atavistic representatives of the clade. The phenomenon is further substantiated by several additional retrogradations that pace the Triassic, a period prone to such events.

Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and 1H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment.

Abstract 1108: B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis

Abstract B-cells, along with other normal and cancerous cells, secrete 50-150 nm vesicles called exosomes. Exosomes carry proteins, mRNA, and miRNA that can be functional upon cellular uptake and are often specific to cell of origin, offering potential as disease biomarkers. Our lab has identified within exosomes from solid tumor cells a cancer-specific protein, Survivin, which is a member of the Inhibitors of Apoptosis (IAP) family. Whether Survivin and other IAP family members such as XIAP, cIAP1 or cIAP2 are also expressed in exosomes from hematopoietic malignancies is yet to be determined. In this study, we aim to investigate the exosomes released from B-cell lymphoma cells and determine the IAP content under basal and sublethal stress conditions. Three human non EBV-transformed B-cell lymphoma lines developed at Wayne State University were used. WSU-FSCCL (follicular small cleaved cell), WSU-DLCL-2 (diffuse large cell), and WSU-WM (Waldenstrom Macroglobulinemia) represent different levels of aggressiveness. Cells were cultured for 24 or 48 hours before exosome isolation from the conditioned media using either ultracentrifugation on a sucrose cushion or ExoQuick (System Biosciences). Sublethal levels of UV irradiation were determined for each cell line by analyzing DNA content by propidium iodide staining with a FACSCalibur. Protein quantity of exosomes was measured using a BCA assay, and protein content was studied using Western blotting. Corresponding gene expression was identified using RT-PCR. Here we report IAP protein and mRNA (except XIAP) within exosomes from basal cells. We are currently probing exosomes from stressed cells for comparison. We hypothesize that the IAP exosomal profile may change under stressed conditions. The specificity and variability in exosomal content offers capabilities not only as disease biomarkers, but also in the immunomodulating activity of exosomes which have shown roles in both stimulating and inhibiting the immune system, We have found evidence that exosomal Survivin plays an immune role in skewing Th1/Th2 populations and decreasing cytotoxic function. Changes in exosome content between basal and stressed cells may have roles in the immunomodulation of the tumor microenvironment which facilitates the evasion of lymphoma cells to immune destruction, as well as possible prognostic benefit as to whether treatments are being effective or detrimental. Citation Format: Heather R. Ferguson Bennit, Malyn M. Asuncion Valenzuela, Jessica S. Jutzy, Nathan R. Wall. B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1108. doi:10.1158/1538-7445.AM2014-1108

Cell Cycle Regulators Guide Mitochondrial Activity in Radiation-Induced Adaptive Response

There are accruing concerns on potential genotoxic agents present in the environment including low-dose ionizing radiation (LDIR) that naturally exists on earth's surface and atmosphere and is frequently used in medical diagnosis and nuclear industry. Although its long-term health risk is being evaluated and remains controversial, LDIR is shown to induce temporary but significant adaptive responses in mammalian cells and animals. The mechanisms guiding the mitochondrial function in LDIR-induced adaptive response represent a unique communication between DNA damage and cellular metabolism. Elucidation of the LDIR-regulated mitochondrial activity may reveal new mechanisms adjusting cellular function to cope with hazardous environmental stress. Key cell cycle regulators, including Cyclin D1/CDK4 and Cyclin B1/cyclin-dependent kinase 1 (CDK1) complexes, are actively involved in the regulation of mitochondrial functions via phosphorylation of their mitochondrial targets. Accumulating new evidence supports a concept that the Cyclin B1/CDK1 complex acts as a mediator in the cross talk between radiation-induced DNA damage and mitochondrial functions to coordinate cellular responses to low-level genotoxic stresses. The LDIR-mediated mitochondrial activity via Cyclin B1/CDK1 regulation is an irreplaceable network that is able to harmonize vital cellular functions with adjusted mitochondrial metabolism to enhance cellular homeostasis. Further investigation of the coordinative mechanism that regulates mitochondrial activities in sublethal stress conditions, including LDIR, will reveal new insights of how cells cope with genotoxic injury and will be vital for future targeted therapeutic interventions that reduce environmental injury and cancer risk.

Enhanced inactivation of Escherichia coli and Listeria monocytogenes by exposure to 405 nm light under sub-lethal temperature, salt and acid stress conditions

The antimicrobial effects of 405nm light have generated interest in its use as an emerging disinfection technology with potential food-related applications. The aim of this study was to assess the bactericidal efficacy of 405nm light for inactivation of Escherichia coli and Listeria monocytogenes under sub-lethally stressed environmental conditions. Bacteria were exposed to 405nm light from a light emitting diode (LED) array under various temperature, salt (NaCl) and acid conditions to determine if bacterial susceptibility to 405nm light inactivation is affected when exposed under these conditions. Non-stressed bacterial populations (105CFU/mL) were exposed to increasing doses of 405nm light (~70mW/cm2) and the inactivation results were compared with those generated under stress conditions. Bacteria were held at various temperatures (4°C, 22°C and 45°C), acid concentrations (pH3, 3.5 and 7) and salt concentrations (0%, 0.8%, 10% and 15% NaCl), and simultaneously exposed to 405nm light. Enhanced inactivation of both E. coli and L. monocytogenes was achieved when light exposure was combined with each of the sub-lethal stresses, with significantly increased inactivation rates compared to non-stressed populations (P≤0.05). One exception was with L. monocytogenes when light-exposed in the presence of 15% salt, as this combination reduced bacterial inactivation. The greatest enhancement of 405nm light inactivation for both bacterial species was achieved when light exposure was combined with sub-lethal acid stress conditions at pH3. This was demonstrated by a 5-log10 reduction of E. coli following a 405nm light dose of 84J/cm2 compared to 378J/cm2 for non-stressed populations (77% reduction in dose) and by a 5-log10 reduction of L. monocytogenes achieved with a dose of 42J/cm2 which corresponded to 50% of the dose required for the equivalent reduction of non-stressed populations. This acid-enhanced 405nm light inactivation effect was demonstrated with E. coli and L. monocytogenes when dispersed in liquid suspension and when deposited on a test surface. Overall, results from this study have shown that sub-lethally stressed bacteria have increased susceptibility to 405nm light inactivation, thereby providing a synergistic inactivation effect, findings which increase the potential of this new light-based decontamination technology for food related applications.

Fast screening of Bifidobacterium longum sublethal stress conditions in a novel two-stage continuous culture strategy

A central issue in the application of probiotics as food additives is their fastidious production and their sensitivity to many environmental stresses. The importance of inducible cell-protective mechanisms triggered by application of sublethal stresses for survival under stress conditions has been demonstrated. Continuous cultures could be a suitable and more efficient method to test stress factors on one culture instead of several repeated batch cultures. In this study, the application of a two-stage continuous culture of Bifidobacterium longum NCC2705 was investigated. The first reactor was operated under fixed conditions at 37 °C and pH 6.0 and used to produce cells with controlled physiology, mimicking cells in the late exponential growth phase. Stress pretreatment combinations of pH (6.0, 5.0 and 4.0), temperature (37, 45 and 47 °C) and NaCl (0, 5 and 10%) were tested in the second reactor. Of all tested combinations, only those of pH 4.0 significantly decreased cell viability in the second reactor compared to control conditions (37 °C, pH 6.0, 0% NaCl) and, therefore, could not be considered as sublethal stresses. Pretreatments with 5 or 10% NaCl had a negative effect on cell viability after gastric lethal stress. A significant improvement in cell resistance to heat lethal stress (56 °C, 5 min) was observed for cells pretreated at 47 °C. In contrast, heat pretreatment negatively affected cell viability after freeze drying and osmotic lethal stresses. The two-stage continuous culture allowed for efficient screening of several stress pretreatments during the same experiment with up to four different conditions tested per day. Optimal sublethal stress conditions can also be applied for producing cells with traditional batch cultures.

Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions

A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.

Polymorphisms in rpoS and Stress Tolerance Heterogeneity in Natural Isolates of Cronobacter sakazakii

Significant phenotypic diversity was observed when we examined the abilities of a number of Cronobacter sakazakii natural isolates to cope with various sublethal stress conditions (acid, alkaline, osmotic, oxidative, or heat stress). Levels of catalase activity and use of acetate as a carbon source, phenotypes commonly used as indirect assays to predict RpoS function, revealed a high correlation between predicted RpoS activity and tolerance to acid, alkaline, osmotic, and oxidative treatments. The rpoS genes were sequenced and analyzed for polymorphisms. Loss-of-function mutations were found in two strains; C. sakazakii DPC 6523 and the genome-sequenced strain C. sakazakii ATCC BAA-894. The complementation of these strains with a functional rpoS gene resulted in an increase in bacterial tolerance to acid, osmotic, and oxidative stresses. The pigmentation status of strains was also assessed, and a high variability in carotenoid content was observed, with a functional rpoS gene being essential for the production of the characteristic yellow pigment. In conclusion, the evidence presented in this study demonstrates that rpoS is a highly polymorphic gene in C. sakazakii, and it supports the importance of RpoS for the tolerance under stress conditions that C. sakazakii may encounter in the food chain and in the host during infection.

Cardiac myocyte‐specific caveolin‐3 overexpression sustains β‐adrenergic receptor response in basal and stressed conditions

Caveolins are scaffolding proteins essential to the formation of caveolae, plasma membrane invaginations that compartmentalize signaling receptors and effector molecules. Cardiac myocyte‐specific overexpression of caveolin‐3 (Cav‐3 OE) increases the number of caveolae in cardiac myocytes. β‐adrenergic receptors (β‐ARs) are localized to caveolae and when stimulated by agonists undergo rapid desensitization to alter cardiac ionotropy and chronotropy. Whether increased expression of Cav‐3 alters β‐AR agonist response in the myocardium is unknown. To test this question, we exposed Langendorff‐perfused hearts from wild‐type (WT) and Cav‐3 OE mice to sublethal ischemic stress (10 min ischemia, 30 min reperfusion) or control conditions and to the β‐AR agonist isoproterenol (iso). In the basal setting, Cav‐3 OE mice had augmented iso‐stimulated left ventricular developed pressure (LVDP) and +dP/dt and also had reduced heart rate relative to WT mice. Following ischemic stress, which desensitizes β‐AR, the difference between Cav‐3 OE and WT mice was even more exaggerated; Cav‐3 OE mice maintained pre‐ischemic LVDP whereas the WT mice had a diminished response. Therefore, modulating expression of Cav‐3 in cardiac myocytes may provide a means to regulate β‐AR responsiveness in the setting of cardiac dysfunction secondary to ischemia.

L-Glutamine inhibits beta-aminobutyric acid-induced stress resistance and priming in Arabidopsis.

The non-protein amino acid beta-aminobutyric acid (BABA) enhances Arabidopsis resistance to microbial pathogens and abiotic stresses through potentiation of the Arabidopsis defence responses. In this study, it is shown that BABA induces the stress-induced morphogenic response (SIMR). SIMR is observed in plants exposed to sub-lethal stress conditions. Anthocyanin, a known modulator of stress signalling, was also found to accumulate in BABA-treated Arabidopsis. These data and a previous microarray study indicate that BABA induces a stress response in Arabidopsis. High concentrations of amino acids, except for L-glutamine, cause a general amino acid stress inhibition. General amino acid inhibition is prevented by the addition of L-glutamine. L-Glutamine was found to inhibit the BABA-mediated SIMR and anthocyanin accumulation, suggesting that the non-protein amino acid BABA causes a general amino acid stress inhibition in Arabidopsis. L-Glutamine also blocked BABA-induced resistance to heat stress and to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. During bacterial infection, priming of the salicylic acid-dependent defence marker PR1 was abolished by L-glutamine treatment. These results indicate that L-glutamine removal of the BABA-mediated stress response is concomitant with L-glutamine inhibition of BABA priming and BABA-induced resistance.

Stress-induced morphogenic responses: growing out of trouble?

Plants exposed to sub-lethal abiotic stress conditions exhibit a broad range of morphogenic responses. Despite the diversity of phenotypes, a generic 'stress-induced morphogenic response' can be recognized that appears to be carefully orchestrated and comprises three components: (a) inhibition of cell elongation, (b) localized stimulation of cell division and (c) alterations in cell differentiation status. It is hypothesized that the similarities in the morphogenic responses induced by distinct stresses, reflect common molecular processes such as increased ROS-production and altered phytohormone transport and/or metabolism. The stress-induced morphogenic response (SIMR) is postulated to be part of a general acclimation strategy, whereby plant growth is redirected to diminish stress exposure.