pH Stress Conditions Research Articles

Evaluation of arsenic phytoremediation potential in Azolla filiculoides Lam. plants under low pH stress conditions

The Azolla filiculoides plants were challenged with different arsenic (As) concentration under low pH stress conditions. The growth rate and doubling time of the plants were severely affected by higher As treatments at pH 5.00 when compared with stress pH 4.75 treatments. Hence, pH 5.00 was considered for further studies. In 10–30 μM As treated cultures, after 6 days, the relative growth rate (RGR) of Azolla plants was significantly reduced and in higher concentration of As, the RGR was negatively regulated. The root trait parameters were also significantly affected by increasing concentrations of As. Further, photosynthetic performance indicators also show significant decline with increasing As stress. Overall, the plants treated with 40 and 50 μM of As displayed stress phenotypes like negative RGR, reduced doubling time and root growth, browning of leaves and root withering. The total proline, H2O2, POD and Catalase activities were significantly affected by As treatments. Meantime, 30 μM of As treated cultures displayed 15 μg/g/Fw As accumulation and moderate growth rate. Thus, the Azolla plants are suitable for the phytoremediation of As (up to 30 μM concentration) in the aquatic environment under low pH conditions (5.00). Furthermore, the transcriptome studies on revealed that the importance of positively regulated transporters like ACR3, AceTr family, ABC transporter super family in As (10 μM) stress tolerance, uptake and accumulation. The transporters like CPA1, sugar transporters, PiT were highly down-regulated. Further, expression analysis showed that the MATE1, CIP31, HAC1 and ACR3 were highly altered during the As stress conditions.

Characterization of Therapeutic Antibody Charge Heterogeneity Under Stress Conditions by Microfluidic Capillary Electrophoresis Coupled with Mass Spectrometry

Therapeutic antibodies are a major class of biopharmaceutics that are applied in disease treatment because of their many advantages, including high specificity and high affinity to molecular targets. Between their production and administration, therapeutic antibodies are exposed to multiple stress conditions. Forced degradation and stress stability studies are conducted to simulate the risk of degradation and the effects of these stresses, thereby enhancing understanding of the drug product to support strategies to mitigate the impact from stressed conditions. These types of studies are also routinely conducted to evaluate product comparability when major process changes are implemented during the production. Charge variant analysis helps understand the changes in the electrostatic environment of biotherapeutics and can uncover underlying molecular level alterations associated with charge variants. Herein, we used ZipChip native capillary electrophoresis-mass spectrometry (nCE-MS) to elucidate the changes in charge variant profiles at the molecular level. In two case studies under thermal stress conditions, we observed that charge variants arose from both post-translational modifications (including deamidation, oxidation, and pyroglutamate formation) and sequence truncations at the hinge regions. Under oxidative stress conditions, oxidation was found to be the major contributor to the changes in the charge variant profiles. Under pH stress conditions, the changes in the charge variant profile were due to increased levels of deamidation, oxidation, and pyroglutamate formation. ZipChip nCE-MS analysis enables identification of charge variant species under various stress conditions, thus supporting process and formulation development of biotherapeutics.

Comparative transcriptome analysis explored the molecular mechanisms of a luxR-type regulator regulating intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila is not a traditional intracellular bacterium. However, previous studies revealed that pathogenic A. hydrophila B11 could temporarily survive for at least 24 h in fish phagocytes, and the regulation of intracellular survival in bacteria was associated with regulators of the LuxR-type. The mechanisms of luxR08110 on the A. hydrophila's survival in macrophages were investigated using comprehensive transcriptome analysis and biological phenotype analysis in this study. The results showed that after luxR08110 was silenced, the intracellular survival ability of bacteria was significantly diminished. Comparative transcriptome analysis revealed that luxR08110 was a critical regulator of A. hydrophila, which regulated the expression of over 1200 genes, involving in bacterial flagellar assembly and chemotaxis, ribosome, sulphur metabolism, glycerolipid metabolism, and other mechanisms. Further studies confirmed that after the inhibition of expression of luxR08110, the motility, chemotaxis and adhesion of A. hydrophila significantly decreased. Moreover, compared with the wild-type strain, the survival rates of silencing strain were all considerably reduced under both H2O2 and low pH stress conditions. According to both transcriptome analysis and phenotypic tests, the luxR08110 of A. hydrophila could act as global regulator in bacteria intracellular survival. This regulator regulated intracellular survival of A. hydrophila mainly through two ways. One way is to regulate bacterial flagellar synthesis and further affects the motility, chemotaxis and adhesion of bacteria. The other way is to regulate sulphur and glycerolipid metabolisms, thus affecting bacterial energy production and the ability to resist environmental stress.

Low pH stress activates several genes for lateral root formation and detoxification of aluminum ions in Cotton plants

In low pH condition of soils (pH<5), Al is solubilized into a phytotoxic form Al(H2O)63+ and inhibits the plant root growth and development. Precisely, during low pH stress (e.g., pH 4.75), aluminum compound converts into its ionic form and becomes higher in level which is a limiting factor for plant root growth and seedling formation. Under aluminum (Al) toxic conditions, the organic acid transporters for example citrate and malate transporters in plasma membrane are involved in organic acid release and form non phytotoxic stable complexes with Al3+ in the rhizosphere. The study showed that, the cotton seedlings exhibited very sensitive phenotypes to low pH stress conditions due to high accumulation of ionic Al stress, the seedlings displayed retarded primary, secondary root growth and small cotyledons. These results indicated that cotton plants are susceptible to low pH condition.. As a whole without fine divisions the root system becomes corraloid in structure which will be having squat secondary roots. Plant has developed a natural defense mechanism in order to combat this unfavorable environment. Expression profile and Al complexation with organic acid exudates studies clearly revealed that both GhALMT1 and GhALS3 genes are involved in Al3+detoxification mechanisms. The remarkable expression was detected for GhALS3 under both low pH and aluminum stress condition in comparison to wild type untreated control whereas GhALMT1 expression was only activated under Al3+ stress condition. Our study, displayed enhanced citrate and malate excretion values in the wild type plants which are treated with Al and H+ stress. Thus, it can be clearly mentioned that GhALMT1 and GhALS3 is playing a significant role in low pHstress tolerance of cotton plant.

PhoP induces RyjB expression under acid stress in Escherichia coli.

Bacterial small RNAs (sRNAs) play a pivotal role in post-transcriptional regulation of gene expression and participate in many physiological circuits. An ~80-nt-long RyjB was earlier identified as a novel sRNA, which appeared to be accumulated in all phases of growth in Escherichia coli. We have taken a comprehensive approach in the current study to understand the regulation of ryjB expression under normal and pH stress conditions. RpoS was not necessary for ryjB expression neither at normal condition nor under acid stress. Hfq also emerged to be unnecessary for RyjB accumulation. Interestingly, RyjB was detected as a novel acid stress induced sRNA. A DNA binding protein PhoP, a component of PhoP/Q regulon, was found to regulate ryjB expression at low pH, as the elimination of phoP allele in the chromosome exhibited a basal level of RyjB expression under acid stress. Ectopic expression of PhoP in ΔphoP cells restored the overabundance of RyjB in the cell. Overexpression of RyjB increased the abundance of sgcA transcripts, with which RyjB shares a 4-nt overlap. The current study increases our knowledge substantially regarding the regulation of ryjB expression in E. coli cell.

Rhizobia inhabiting &lt;em&gt;Clitoria ternatea&lt;/em&gt; L. in Anuradhapura District, Sri Lanka: An assessment of stress tolerance and genetic diversity

Legume-Rhizobial symbiosis plays a greater agronomical and ecological significance as it provides fixed nitrogen through Biological Nitrogen Fixation. <em>Clitoria ternatea </em>is a leguminous plant that hosts a wide range of rhizobial strains. Even though it is a widely distributed plant, comprehensive information and studies conducted on the rhizobial - <em>C.</em> <em>ternatea </em>symbiosis is lacking in Sri Lanka. This study aimed to identify different stress-tolerant rhizobial strains inhabiting root nodules of <em>C.</em> <em>ternatea </em>growing in seven selected locations of Anuradhapura district of Sri Lanka<em>. </em>Twenty-eight pure rhizobial colonies were isolated and they were separately grown in ½ Lupin broths and were subjected to four different physiological conditions, pH, temperature, salinity, and drought. Most of the isolates were well-grown within the pH range of 5.0-8.0, the temperature range of 30-35°C and at 0.2% Polyethylene glycol-8000 (PEG) concentration. There was no observable pattern in the growth of rhizobial strains in different physiological conditions. The twelve rhizobial strains which showed high tolerance to extreme physiological conditions were subjected to a combination of physiological stress conditions of pH 8.0, temperature 36°C, 3.0 % NaCl and 0.4 % PEG. The maximum growth in combination physiological study was observed in a strain collected at the Palugaswewa site. According to the dendrogram prepared by the Enterobacterial Repetitive Intergenic Consensus (ERIC) profile, twelve strains are genetically diverse, as they belonged to11 clusters at 69.89 % of similarity level. These stress-tolerant rhizobial strains could be used for further studies on cross-inoculation of crop legumes as an alternative to the nitrogenous fertilizers.

Recent advances in engineering of microbial cell factories for intelligent pH regulation and tolerance.

pH regulation is a serious concern in the industrial fermentation process as pH adjustment heavily utilizes acid/base and pollutes the environment. Under pH-stress conditions, microbial growth and production of valuable target products may be severelyaffected. Furthermore, some strains generating acidic or alkaline products require self pH regulation and increased tolerance against pH-stress. For pH control, synthetic biology has provided advanced engineering approaches to construct robust and more intelligent microbial strains, exhibiting tolerance to pH-stress to cope with limitations of pH regulation. This study reviewed the current progress of advanced strain evolution strategies to engineer pH-stress tolerant strains via synthetic biology. In addition, a large number of pH-responsive elements, including promoters, riboswitches, and some proteins have been investigated and applied for construction of pH-responsive genetic circuits and intelligent pH-responsive microbial strains.

Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli

Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D ≥μmax of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35−0.37 mg L−1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

Speeding up the Development of 5-[(4-Chlorophenoxy)-Methyl]-1,3,4-Oxadiazole-2-Thiol as Successful Oral Drug Candidate Based on Physicochemical Characteristics

The present paper describes for the first time the oral-drug-qualifying predictors, anti-acetylcholinesterase activity, and molecular docking of a new compound, 5-[(4-chlorophenoxy)-methyl]-1,3,4-oxadiazole-2-thiol. The oral bioavailability predictors such as lipophilicity (logP), ionization constant (pKa), solubility (logS) and permeability (logD) were predicted using computational tools. Then, a UV spectrophotometric method was developed to verify the predicted properties and determine stability of the proposed compound in the presence of various stressors. The predicted logP and pKa values were very close to the measured values, while, the computed aqueous solubility (6.46 μg/mL) was lower than that found experimentally (65.00 μg/mL). The logS versus pH and logD versus pH plots indicated that the compound migrated to hydrophilic compartment on increasing pH. The compound remained stable under various photolytic and pH stress conditions. However, the compound exhibited degradation under oxidative and thermal stress. The results of the present study indicate that the proposed compound absorbs well from the oral route and remains stable at ambient temperature and physiological pH. The observed remarkable dose-dependent anti-acetylcholinesterase activity indicates that the compound may be a drug candidate for treating neurodegenerative disorders.

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

AbstractBACKGROUNDRecombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a‐pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high‐level soluble pod production.RESULTSDuring recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L−1 at pH 6.0 after 64 h induction.CONCLUSIONSoluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance. © 2019 Society of Chemical Industry

Variable Carbon Source Utilization, Stress Resistance, and Virulence Profiles Among Listeria monocytogenes Strains Responsible for Listeriosis Outbreaks in Switzerland.

A combination of phenotype microarrays, targeted stress resistance and virulence assays and comparative genome analysis was used to compare a set of Listeria monocytogenes strains including those involved in previous Swiss foodborne listeriosis outbreaks. Despite being highly syntenic in gene content these strains showed significant phenotypic variation in utilization of different carbon (C)-sources as well as in resistance of osmotic and pH stress conditions that are relevant to host and food associated environments. An outbreak strain from the 2005 Swiss Tomme cheese listeriosis outbreak (Lm3163) showed the highest versatility in C-sources utilized whereas the strain responsible for the 1983 to 1987 Vacherin Montd’or cheese listeriosis outbreak (LL195) showed the highest tolerance to both osmotic and pH stress conditions among the examined strains. Inclusion of L-norvaline led to enhanced resistance of acidic stress in all the examined strains and there were strain–strain-specific differences observed in the ability of other amino acids and urea to enhance acid stress resistance in L. monocytogenes. A strain dependent inhibition pattern was also observed upon inclusion of β-phenylethylamine under alkaline stress conditions. In targeted phenotypic analysis the strain-specific differences in salt stress tolerance uncovered in phenotypic microarrays were corroborated and variations in host cell invasion and virulence among the examined strains were also revealed. Outbreak associated strains representing lineage I serotype 4b showed superior pathogenicity in a zebrafish infection model whilst Lm3163 a lineage II serotype 1/2a outbreak strain demonstrated the highest cellular invasion capacity amongst the tested strains. A genome wide sequence comparison of the strains only revealed few genetic differences between the strains suggesting that variations in gene regulation and expression are largely responsible for the phenotypic differences revealed among the examined strains. Our results have generated data that provides a potential basis for the future design of improved Listeria specific media to enhance routine detection and isolation of this pathogen as well as provide knowledge for developing novel methods for its control in food.

Driving Factors of the Dynamics of Microbial Community in a Dam of Copper Mine Tailings

The relative importance of the deterministic versus stochastic processes underlying community dynamics has long been a central theme in community ecology, and is intensively debated in the field. Microbial communities play key roles in nutrient cycling and the flow of energy in ecosystems. The research on the structural dynamics of microbial community will provide data and theoretical support for understanding the assembly mechanisms of community, and for predicting the dynamics of microbial community under environmental stress. In this study, the Illumina MiSeq method was applied to investigate the structural dynamics of bacterial and fungal community in a dam of Shibahe mine tailings at different restoration stages (1-45 years). The results indicated that the soil physicochemical properties in the dam of mine tailings formed an ecological gradient, and the plant community showed succession along the restoration time. The diversity of plant communities was significantly correlated with soil nutrient contents but not with soil heavy metal contents. The structure of the microbial communities showed significant differences at different restoration stages of the dam land, in which Proteobacteria, Actinobacteria, Firmicutes, and Acidobacteria were the dominant bacterial phyla, and Ascomycota, Basidiomycota, and Zygomycota were the dominant fungal phyla. The assembly of the microbial community was shaped mainly by the soil nutrients and soil heavy metal contents, but plant diversity had no significant effect on the microbial community structure. It was suggested that edaphic factors drive the dynamics of microbial communities under the stress conditions of pH and heavy metals on small, local scales.

Growth and norharmane production of Chroococcus minutus under various stress conditions

Cyanobacterium samples were collected from fresh water of Tokat city in Turkey, and then isolation and cultivation of Chroococcus minutus were achieved successfully. TLC (Thin layer chromatography) and HPLC (High Performance Liquid Chromatography) analyses revealed that the C. minutus consisted of norharmane as a major product. So amount of norharmane was determined during the growth process. Growth and norharmane production of C. minutus were executed under salt stress and pH stress conditions. The most growth and the highest production of norharmane were detected at 16th day. Therefore inoculation process was performed at 16th day. Salt stress was evaluated at 0.5, 1.0, 3.0 and 5.0 M concentrations. The most norharmane was synthesized by C. minutus at 5 M concentration. The norharmane production and the growth were higher at pH 9 than that of the pH 5. Most norharmane was produced at pH 7.

Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies

Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products.

A Network Biology Approach to Decipher Stress Response in Bacteria UsingEscherichia coliAs a Model

The development of drug-resistant pathogenic bacteria poses challenges to global health for their treatment and control. In this context, stress response enables bacterial populations to survive extreme perturbations in the environment but remains poorly understood. Specific modules are activated for unique stressors with few recognized global regulators. The phenomenon of cross-stress protection strongly suggests the presence of central proteins that control the diverse stress responses. In this work, Escherichia coli was used to model the bacterial stress response. A Protein-Protein Interaction Network was generated by integrating differentially expressed genes in eight stress conditions of pH, temperature, and antibiotics with relevant gene ontology terms. Topological analysis identified 24 central proteins. The well-documented role of 16 central proteins in stress indicates central control of the response, while the remaining eight proteins may have a novel role in stress response. Cluster analysis of the generated network implicated RNA binding, flagellar assembly, ABC transporters, and DNA repair as important processes during response to stress. Pathway analysis showed crosstalk of Two Component Systems with metabolic processes, oxidative phosphorylation, and ABC transporters. The results were further validated by analysis of an independent cross-stress protection dataset. This study also reports on the ways in which bacterial stress response can progress to biofilm formation. In conclusion, we suggest that drug targets or pathways disrupting bacterial stress responses can potentially be exploited to combat antibiotic tolerance and multidrug resistance in the future.

Growth rate analysis and protein identification of Kappaphycus alvarezii (Rhodophyta, Gigartinales) under pH induced stress culture

Environmental pH is one of the factors contributing to abiotic stress which in turn influences the growth and development of macroalgae. This study was conducted in order to assess the growth and physiological changes in Kappaphycus alvarezii under different pH conditions: pHs 6, ∼8.4 (control) and 9. K. alvarezii explants exhibited a difference in the daily growth rate (DGR) among the different pH treatments (p≤0.05). The highest DGR was observed in control culture with pH ∼8.4 followed by alkaline (pH 9) and acidic (pH 6) induced stress cultures. Protein expression profile was generated from different pH induced K. alvarezii cultures using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein identification and analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and Mascot software. Ribulose bisphosphate carboxylase (Rubisco) large chain was identified to be up-regulated under acidic (pH 6) condition during the second and fourth week of culture. The findings indicated that Rubisco can be employed as a biomarker for pH induced abiotic stress. Further study on the association between the expression levels of Rubisco large chain and their underlying mechanisms under pH stress conditions is recommended.

Enhancement of thiamine release during synthetic mutualism between Chlorella sorokiniana and Azospirillum brasilense growing under stress conditions

Thiamine release during synthetic mutualism between Chlorella sorokiniana co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense was measured under stress conditions of pH, light intensity, and nitrogen starvation in short-term experiments. Thiamine release in the co-immobilized treatment was significantly higher at acidic pH compared to thiamine released by either microorganism alone. Under slightly alkaline pH, C. sorokiniana released the highest amount of thiamine. At stressful pH 6, the co-immobilized treatment released a higher quantity of thiamine than the sum of thiamine released by either microorganisms when immobilized separately. Release of thiamine by C. sorokiniana alone or co-immobilized was light intensity dependent; with higher the light intensity, more thiamine was released. Extreme light intensity negatively affected growth of the microalgae and release of thiamine. Nitrogen starvation during the first 24 h of culturing negatively affected release of thiamine by both microorganisms, where C. sorokiniana was more severely affected. Partial or continuous nitrogen starvation had similar negative effects on C. sorokiniana, but co-immobilization improved thiamine release. These results indicate that thiamine is released during synthetic mutualism between C. sorokiniana and A. brasilense, and this happens specifically during the alleviation of pH stress in the microalgae.

CDNA cloning and expression profile analysis of an ATP-binding cassette transporter in the hepatopancreas and intestine of shrimp Fenneropenaeus chinensis

ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2122-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Fenneropenaeus chinensis. The deduced protein, FcABCG, contains 633 amino acid residues with a theoretical molecular weight of 70.57kDa. Phylogenetic analysis showed that the FcABCG clusters with the shrimp Litopenaeus vannamei ABCG in the same clade and is more closely related to the insect ABC transporter/white proteins than to the ABCG2 of the fish Salmo salar. High-level mRNA expression of FcABCG was detected in the tissues of hepatopancreas and intestine using a real-time quantitative reverse transcriptase PCR. Furthermore, exposure to alkaline pH stress conditions and norfloxacin diets result in the activation of the FcABCG gene in F. chinensis. These results suggest that the ABCG transporter of F. chinensis might play a role in coping high pH stress and in exporting xenobiotics.

Use of CE‐SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress

CE-SDS gel technique has been used extensively in the field of monoclonal antibody (mAb) as a tool for product purity, stability, and characterization. It offers many advantages over the traditional labor-intensive SDS-PAGE slab gel technology with respect to speed and resolution. Monoclonal antibodies are known to cleave in the hinge region due to extreme pH, high temperature and in the presence of metals, especially copper. This cleavage will impact the shelf lifetime of mAb product hence its quality. CESDS gel method using Beckman PA800 with UV detection is used to characterize the effects of copper and other metals such as iron and zinc on mAb clipping. In addition, mAb integrity under high temperature and high pH stress conditions was also evaluated and the results clearly show that CE-SDS gel can distinguish clipping due to copper versus heat and/or high pH. The data presented illustrate the power of this simple CESDS gel technique in supporting the development of mAb from product quality and stability to the final product characterization.

Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes

To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45 degrees C), NaCl (5.5%), oxidative stress (15 mmol(-1) H2O2), acidic pH (5.5) and ethanol (5%) for 3 h (short-term stress) or for 5 days (long-term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12-16% reductions in reaction for anti-E. coli O157:H7 and 20-48% reductions for anti-Salmonella polyclonal antibodies during short-term stress, whereas the most stresses exhibited enhanced reaction (44-100% increase) with the anti-L. monocytogenes polyclonal antibody. During long-term stress exposure to combined stress conditions of pH 5.5, 3.5% NaCl at 12 degrees C or at 4 degrees C, antibody reactions to the three pathogens were highly variable with the combined stress at 4 degrees C showing the most reductions (8-40%). Likewise, there were about 18-59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short- and long-term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. Overall, the antibody reactions were significantly reduced in pathogens exposed to both short- and long-term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.