Active Acid Research Articles

Effect of ultrasound combined with malic acid on the activity and conformation of mushroom (Agaricus bisporus) polyphenoloxidase.

Polyphenoloxidase (PPO) plays an important role in the browning of vegetables, fruits and edible fungi. The effects of ultrasound, malic acid, and their combination on the activity and conformation of mushroom (Agaricus bisporus) PPO were studied. The activity of PPO decreased gradually with the increasing of malic acid concentrations (5-60mM). Neither medium concentrations (10, 20, 30mM) malic acid nor individual ultrasound (25kHz, 55.48W/cm(2)) treatment could remarkably inactivate PPO. However, the inactivation during their combination was more significant than the sum of ultrasound inactivation and malic acid inactivation. The inactivation kinetics of PPO followed a first-order kinetics under the combination of ultrasound and malic acid. The conformation of combination treated PPO was changed, which was reflected in the decrease of α-helix, increase of β-sheet contents and disruption of the tertiary structure. Results of molecular microstructure showed that ultrasound broke large molecular groups of PPO into small ones. Moreover, combined treatment disrupted the microstructure of PPO and molecules were connected together.

Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion.

Heterosis has been widely used in agriculture, but the underlying molecular principles are still largely unknown. During seed germination, we observed that maize (Zea mays) hybrid B73/Mo17 was less sensitive than its parental inbred lines to exogenous abscisic acid (ABA), and endogenous ABA content in hybrid embryos decreased more rapidly than in the parental inbred lines. ZmABA8ox1b, an ABA inactivation gene, was consistently more highly up-regulated in hybrid B73/Mo17 than in its parental inbred lines at early stages of seed germination. Moreover, ectopic expression of ZmABA8ox1b obviously promoted seed germination in Arabidopsis Remarkably, microscopic observation revealed that cell expansion played a major role in the ABA-mediated maize seed germination heterosis, which could be attributed to the altered expression of cell wall-related genes.

The TCA cycle transferase DLST is important for MYC-mediated leukemogenesis.

Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset in zebrafish without detectable effects on fish development. DLST is the E2 transferase of the α-ketoglutarate (α-KG) dehydrogenase complex (KGDHC), which converts α-KG to succinyl-CoA in the TCA cycle. RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of α-KG and a decrease of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Together, our studies uncovered an important role for DLST in MYC-mediated leukemogenesis and demonstrated the metabolic dependence of T-lymphoblasts on the TCA cycle, thus providing implications for targeted therapy.

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142→R or H142→F or H142→A substitutions resulted in non-infectious FMDV, H142→D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

Inactivation and Disinfection of Zika Virus on a Nonporous Surface

The zika virus (ZIKV), an emergent arbovirus within the flavivirus family, is of public health concern due to the lack of a vaccine and possibility of teratogenic effects in infected pregnant women. The flaviviruses are enveloped, and it might be expected that inactivation methods for enveloped viruses in general might be effective for ZIKV. We have investigated a number of physical and chemical inactivation approaches using both low protein (5% serum) and 90% blood organic loads present during drying of virus onto carriers. ZIKV in 90% blood displayed ~0.06 log10 per hour inactivation over 8 h, while virus in 5% serum was inactivated at a much higher rate (~0.5 log10 per h). ZIKV was susceptible to dry heat treatment (56°C–60°C) when dried in 5% serum, but less so when dried in 90% blood. A quaternary ammonium/alcohol-based product and 70% isopropyl alcohol caused complete (>3.5 and >5 log10, respectively) inactivation of ZIKV in 15 s regardless of the organic load. Efficacy of inactivation of ZIKV by chlorine was highly dependent on the organic load at time of drying, with complete (>4 log10) inactivation being observed in 15 s by 500 ppm chlorine at 5% serum. Inactivation in the presence of 90% blood required 5,000 ppm chlorine to achieve >2 log10 inactivation, and 10,000 ppm chlorine to achieve >3 log10. Peracetic acid (1,000 ppm) inactivation also displayed a striking dependence on organic load, with complete (>4 log10) inactivation observed in 15 s at 5% serum and <1.5 log10 reduction in 5 minutes in a 90% blood matrix. When suspended in solutions of pH 4.0 or pH 10.0 at time of drying, ZIKV displayed <1.5 log10 reduction in 5 min, regardless of organic load. In conclusion, ZIKV displays susceptibility to commonly employed disinfectants similar to that of other flaviviruses.

БИОКАТАЛИТИЧЕСКИЕ СВОЙСТВА ИММОБИЛИЗОВАННОЙ ИНУЛИНАЗЫ

The heterogeneous biocatalyst based on inulinasе immobilized on the non-ionogenic sorbent of Stirosorb was proposed. Investigation of thermic and acid inactivation of free and immobilized inulinasе was carried out. The corresponding values of the inactivation constants were rated. Increase of thermal stability of the immobilized ferment compared to the free one was exposed. Possibility of use of the immobilized ferment ten cycles long during the reaction of inulin hydrolysis was determined.

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus.

Infection by foot-and-mouth disease virus (FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease (FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements (VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

Evaluating Efficacy of Field-Generated Electrochemical Oxidants on Disinfection of Fomites Using Bacteriophage MS2 and Mouse Norovirus MNV-1 as Pathogenic Virus Surrogates

Surface disinfection, as part of environmental hygiene practices, is an efficient barrier to gastroenteritis transmission. However, surface disinfectants may be difficult to obtain in remote, resource-limited, or disaster relief settings. Electrochemical oxidants (ECO) are chlorine-based disinfectants that can be generated using battery power to electrolyze brine (NaCl) solutions. Electrolysis generates a mixed-oxidant solution that contains both chlorine (HOCl, OCl(-)) and reactive oxygen species (e.g., ·OH, O3, H2O2, and ·O2-) capable of inactivating pathogens. One onsite generator of ECO is the Smart Electrochlorinator 200 (SE-200, Cascade Designs, Inc.). In a laboratory study, we assessed ECO surface disinfection efficacy for two gastrointestinal virus surrogates: bacteriophage MS2 and murine norovirus MNV-1. We quantified both infectivity and nucleic acid inactivation using culture-dependent and independent assays. At free available chlorine concentrations of 2,500ppm and a contact time of 30s, ECO inactivation of infective MS2 bacteriophage exceeded 7 log10 compared to MNV-1 disinfection of approximately 2 log10. Genomic RNA inactivation was less than infective virus inactivation: MS2 RNA inactivation was approximately 5 log10 compared to MNV-1 RNA inactivation of approximately 1.5 log10. The results are similar to inactivation efficacy of household bleach when used at similar free available chlorine concentrations. Our work demonstrates the potential of ECO solutions, generated onsite, to be used for surface disinfection.

Specific growth rate determines the sensitivity of Escherichia coli to lactic acid stress: implications for predictive microbiology.

This study tested the hypothesis that sensitivity of Escherichia coli to lactic acid at concentrations relevant for fermented sausages (pH 4.6, 150 mM lactic acid, a w = 0.92, temperature = 20 or 27°C) increases with increasing growth rate. For E. coli strain 683 cultured in TSB in chemostat or batch, subsequent inactivation rates when exposed to lactic acid stress increased with increasing growth rate at harvest. A linear relationship between growth rate at harvest and inactivation rate was found to describe both batch and chemostat cultures. The maximum difference in T90, the estimated times for a one-log reduction, was 10 hours between bacteria harvested during the first 3 hours of batch culture, that is, at different growth rates. A 10-hour difference in T90 would correspond to measuring inactivation at 33°C or 45°C instead of 37°C based on relationships between temperature and inactivation. At similar harvest growth rates, inactivation rates were lower for bacteria cultured at 37°C than at 15–20°C. As demonstrated for E. coli 683, culture conditions leading to variable growth rates may contribute to variable lactic acid inactivation rates. Findings emphasize the use and reporting of standardised culture conditions and can have implications for the interpretation of data when developing inactivation models.

Selection and characterization of an acid-resistant mutant of serotype O foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.

Enhanced inactivation of Salmonella Typhimurium from blueberries by combinations of sodium dodecyl sulfate with organic acids or hydrogen peroxide

Increasing consumption of raw blueberries has led to the need for improved food safety in the berry fruit industry. This study was undertaken to evaluate an enhanced Salmonella inactivation on blueberries by washing with sodium dodecyl sulfate (SDS) in combination with one of common antimicrobial agents (chlorine, lactic acid, acetic acid, citric acid and hydrogen peroxide). In addition, the impacts of most effective treatments on apparent quality, total phenolic, anthocyanin content, mold and yeast, and Salmonella survivors during the storage of blueberries were also determined. Maximum reductions of Salmonella after washing with 0.5mg/ml acetic acid plus 5000ppm SDS and 200ppm hydrogen peroxide containing 5000ppm SDS were 4.0 and 4.2log10CFU/g, respectively. Addition of SDS enhanced Salmonella inactivation of the acetic acid or hydrogen peroxide solutions. These treatments showed similar Salmonella reductions with 200ppm chlorine (P>0.05). None of these washings decreased the total phenolic, anthocyanin content and apparent quality. Following the two effective antimicrobial washes, 0.5mg/ml acetic acid plus 5000ppm SDS and 200ppm hydrogen peroxide containing 5000ppm SDS, populations of Salmonella on blueberries remained constant throughout the 3-day storage with no appreciable growth. Both treatments decreased counts of yeasts and molds throughout the storage. Therefore, the use of 0.5mg/ml acetic acid plus 5000ppm SDS, 200ppm hydrogen peroxide in combination with 5000ppm SDS may have practical potential as an alternative to the use of chlorine-based washing solution for blueberries.

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.

Evaluation of disinfection efficacy of performic acid (PFA) catalyzed by sulfuric and ascorbic acids tested on Escherichia coli (ATCC, 8739)

Performic acid (PFA) is an oxidizing agent which has recently attracted attention its for use as disinfectant in the medical field and food industry. This study investigated the efficacy of PFA catalyzed by both sulfuric and ascorbic acid. Tap water in one experiment and sterilized activated sludge effluent (ASE) in another, were used for the preparation of aqueous solutions. Escherichia coli was used as the target micro-organism to investigate the efficacy of catalyzed PFA. The results were analyzed to set up the pertinent mathematical relationship according to Hom’s model. Applying the highest initial dose of 15 mg l−1, showed the superior catalytic action of sulfuric acid and 7-log inactivation was achieved in 30 min of disinfection period. Application of PFA on E. coli in our experiment caused CT value of 12.16 mg min l−1 for 99% inactivation and CT value of 23.82 mg min l−1 for 99.9% inactivation. PFA produced 4-log inactivation at a CT value of 35.48 mg min l−1. The results showed that, the promoting action of sulfuric acid is expected in higher initial dose of PFA when synthetic liquid medium was prepared of sterilized ASE. The disinfection efficacy of PFA is proved to be less than free chlorine and more than chloramines.

The rice GERMINATION DEFECTIVE 1, encoding a B3 domain transcriptional repressor, regulates seed germination and seedling development by integrating GA and carbohydrate metabolism

It has been shown that seed development is regulated by a network of transcription factors in Arabidopsis including LEC1 (LEAFY COTYLEDON1), L1L (LEC1-like) and the B3 domain factors LEC2, FUS3 (FUSCA3) and ABI3 (ABA-INSENSITIVE3); however, molecular and genetic regulation of seed development in cereals is poorly understood. To understand seed development and seed germination in cereals, a large-scale screen was performed using our T–DNA mutant population, and a mutant germination-defective1 (gd1) was identified. In addition to the severe germination defect, the gd1 mutant also shows a dwarf phenotype and abnormal flower development. Molecular and biochemical analyses revealed that GD1 encodes a B3 domain-containing transcription factor with repression activity. Consistent with the dwarf phenotype of gd1, expression of the gibberelic acid (GA) inactivation gene OsGA2ox3 is increased dramatically, accompanied by reduced expression of GA biosynthetic genes including OsGA20ox1, OsGA20ox2 and OsGA3ox2 in gd1, resulting in a decreased endogenous GA4 level. Exogenous application of GA not only induced GD1 expression, but also partially rescued the dwarf phenotype of gd1. Furthermore, GD1 binds to the promoter of OsLFL1, a LEC2/FUS3-like gene of rice, via an RY element, leading to significant up-regulation of OsLFL1 and a large subset of seed maturation genes in the gd1 mutant. Plants over-expressing OsLFL1 partly mimic the gd1 mutant. In addition, expression of GD1 was induced under sugar treatment, and the contents of starch and soluble sugar are altered in the gd1 mutant. These data indicate that GD1 participates directly or indirectly in regulating GA and carbohydrate homeostasis, and further regulates rice seed germination and seedling development.

Relationship between gene responses and symptoms induced by Rice grassy stunt virus

Rice grassy stunt virus (RGSV) is a serious threat to rice production in Southeast Asia. RGSV is a member of the genus Tenuivirus, and it induces leaf yellowing, stunting, and excess tillering on rice plants. Here we examined gene responses of rice to RGSV infection to gain insight into the gene responses which might be associated with the disease symptoms. The results indicated that (1) many genes related to cell wall synthesis and chlorophyll synthesis were predominantly suppressed by RGSV infection; (2) RGSV infection induced genes associated with tillering process; (3) RGSV activated genes involved in inactivation of gibberellic acid and indole-3-acetic acid; and (4) the genes for strigolactone signaling were suppressed by RGSV. These results suggest that these gene responses to RGSV infection account for the excess tillering specific to RGSV infection as well as other symptoms by RGSV, such as stunting and leaf chlorosis.

Is it all about Contact? Neurodegeneration as a “Protein Freeze Tag Game” Inside the Central Nervous System

Is it all about Contact? Neurodegeneration as a “Protein Freeze Tag Game” Inside the Central Nervous System

An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation

The aim of this study was to define an integrative approach to identify resistance biomarkers using gene expression quantification and mathematical modelling. Mid-exponentially growing cells were transferred into acid conditions (BHI, pH 4.6) to obtain inactivation kinetics, performed in triplicate. The inactivation curve was fitted with a mixed Weibull model. This model allowed to differentiate two subpopulations with various acid resistances among the initial population. In parallel, differential gene expression was quantified by RT-qPCR. While narL was down-regulated throughout acid inactivation, sigB and katA were up-regulated. sigB expression up-regulation peak was correlated to the less resistant subpopulation when katA up-regulation, was correlated to the more resistant subpopulation. Moreover, differences in population structure were highlighted between each replicate. The higher proportion of the more resistant subpopulation was linked to a higher katA gene expression. These results suggest that sigB and katA might be used as different types of biomarkers, for instance to track moderate and high acid-resistance, respectively. The use of this approach combining RT-qPCR and predictive modelling to track cellular biomarker variations appears as an interesting tool to take into account physiological cell responses into mathematical modelling, allowing an accurate prediction of microbial behaviour.

Comparing inactivation protocols of Yersinia organisms for identification with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

It is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined. Here, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ <0.001, ANOVA test). The signal-to-noise ratio for the m/z 6049 peak present in all of the tested Yersinia isolates was 9.7 ± 3.1 for protocol A and 18.1 ± 4.6 for protocol B (ρ < 0.001). Compared with spectra in our local database containing 48 Yersinia spp., including 20 strains of Y. pestis, the identification score was 1.79 ± 0.2 for protocol A and 1.97 ± 0.19 for protocol B (ρ = 0.0024). Our observations indicate that for the identification of Yersinia organisms, ethanol inactivation yielded MALDI-TOF-MS spectra of significantly higher quality than spectra derived from trifluoroacetic acid inactivation. Combined with previously published data, our results permit the updating of protocols for inactivating BSL-3 bacteria.

Enhanced expression of retinoic acid-metabolizing enzyme CYP26A1 in sunlight-damaged human skin

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis. CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, has been shown to result in a state of functional VAD of the cell. Recently, we demonstrated that CYP26A1 efficiently promotes cell survival properties and eventually contributes to the carcinogenic process, implying roles as an oncogene. To clarify the possible association between VAD caused by CYP26A1 expression and the development of human epithelial neoplasia, we examined whether enhanced expression of CYP26A1 might be observed in various lesions of human skin. We report here that basal keratinocytes showed only weak positivity of CYP26A1 in sunlight-nonexposed areas, whereas strong positive staining was observed in skin from chronically sunexposed body areas and in epidermis that had the dysplastic changes known as actinic keratosis. However, we found no expression of constitutive CYP26A1 in skin malignancies such as squamous cell carcinomas. Our observation suggests an involvement of enhanced CYP26A1 expression causing a functional VAD state in skin that can potentially lead to neoplastic transformation of keratinocytes in an early phase during skin carcinogenesis.

Identification of transcriptome profiles and signaling pathways for the allelochemical juglone in rice roots

Juglone (5-hydroxy-1,4-naphthoquinone) is known allelochemical, but its molecular mode of action is not well understood. We found that juglone induced reactive oxygen species production and calcium accumulation. To gain more insight into these cellular responses, we performed large-scale analysis of the rice transcriptome during juglone stress. Exposure to juglone triggered changes in transcript levels of genes related to cell growth, cell wall formation, chemical detoxification, abiotic stress response and epigenesis. The most predominant transcription-factor families were AP2/ERF, HSF, NAC, C2H2, WRKY, MYB and GRAS. Gene expression profiling of juglone-treated rice roots revealed upregulated signaling and biosynthesis of abscisic acid and jasmonic acid and inactivation of gibberellic acid. In addition, juglone upregulated the expression of two calcium-dependent protein kinases (CDPKs), 6 mitogen-activated protein kinase (MAPK) genes and 1 MAPK gene and markedly increased the activities of a CDPK-like kinase and MAPKs. Further characterization of these juglone-responsive genes may be helpful for better understanding the mechanisms of allelochemical tolerance in plants.