Main Inactivation Mechanism Research Articles

Inactivation mechanism of E. coli in water by enhanced photocatalysis under visible light irradiation

Developing efficient and economical technologies for drinking water disinfection remains a challenge. We synthesized Ag/AgBr/LDH doped with various silver mass concentrations and explored its ability to inactivate E. coli under visible light irradiation (λ ≥ 400 nm). Our results indicated a total inactivation of E. coli (107 CFU·mL−1) within 80 min using 2 % Ag/AgBr/LDH in a laboratory-scale test. The method was evaluated for disinfecting three effluent samples collected from one drinking water treatment plant, covering representative water treatment processes. After five consecutive runs, the inactivation efficiency decreased slightly to 89 % in CFU·mL−1, indicating that the photocatalysts had excellent stability and reusability. The mechanisms were analyzed by combining chemical and biological methods. It was verified that singlet oxygen (1O2), hydrogen peroxide (H2O2), and photo-generated electrons (e−) were significant contributors to the inactivation process. Scanning electron microscopy images analysis showed the disruption of the membrane integrity of E. coli by photocatalytic oxidation. Internal component leakage and reduced enzyme activity were also observed in terms of K+ leakage, β-galactosidase activity, and antioxidant enzyme activity. The results by the transcriptomic analysis implied that Ag/AgBr/LDH regulating the oxidative stress response and cell membrane damage related genes was the main inactivation mechanism.

Exploration of reaction rates of chlorine dioxide with tryptophan residue in oligopeptides and proteins

Chlorine dioxide (ClO2), an alternative disinfectant to chlorine, has a superior ability to inactivate microorganisms, in which protein damage has been considered as the main inactivation mechanism. However, the reactivity of ClO2 with amino acid residues in oligopeptides and proteins remains poorly investigated. In this research, we studied the reaction rate constants of ClO2 with tryptophan residues in five heptapeptides and four proteins using stopped-flow or competition kinetic method. Each heptapeptide and protein contain only one tryptophan residue and the reactivity of tryptophan residue with ClO2 was lower than that of free tryptophan (3.88 × 104 (mol/L)−1sec−1 at pH 7.0). The neighboring amino acid residues affected the reaction rates through promoting inter-peptide aggregation, changing electron density, shifting pKa values or inducing electron transfer via redox reactions. A single amino acid residue difference in oligopeptides can make the reaction rate constants differ by over 60% (e.g. 3.01 × 104 (mol/L)−1sec−1 for DDDWNDD and 1.85 × 104 (mol/L)−1sec−1 for DDDWDDD at pH 7.0 (D: aspartic acid, W: tryptophan, N: asparagine)). The reaction rates of tryptophan-containing oligopeptides were also highly pH-dependent with higher reactivity for deprotonated tryptophan than the neutral specie. Tryptophan residues in proteins spanned a 4-fold range reactivity toward ClO2 (i.e. 0.84 × 104 (mol/L)−1sec−1 for ribonuclease T1 and 3.21 × 104 (mol/L)−1sec−1 for melittin at pH 7.0) with accessibility to the oxidant as the determinating factor. The local environment surrounding the tryptophan residue in proteins can also accelerate the reaction rates by increasing the electron density of the indole ring of tryptophan or inhibit the reaction rates by inducing electron transfer reactions. The results are of significance in advancing understanding of ClO2 oxidative reactions with proteins and microbial inactivation mechanisms.

A slurry microcosm study on the interaction between antibiotics and soil bacterial community

Antibiotics released in the environment have attracted great attention. The environmental emission control of antibiotics should be based on the degree of their negative impacts on the environment and ecology. Here, we conducted a series of soil slurry microcosm experiments to investigate the interactions between antibiotics and the soil bacterial community. In the soil slurry, distinctive behaviors were observed for different antibiotics. Beta-lactams (ampicillin and ceftriaxone) experienced fast biodegradation. Kanamycin was adsorbed on soil particles soon after its addition. Nalidixic acid was stable throughout the experimental period (164 h). The main inactivation mechanism of tetracycline was deduced to be hydrolysis. Bacterial communities in slurries with or without antibiotic-treatment were profiled via high-throughput Illumina sequencing of the 16S rRNA gene. Unstable (ceftriaxone) and adsorbed (kanamycin) antibiotics show minor or negligible influences on the soil bacterial community. Stable antibiotics (nalidixic acid and tetracycline) have significantly affected the structure of the bacterial community. Most of enriched bacterial genera by various antibiotics belong to the same phylum, Proteobacteria. Inhibited bacterial phyla by nalidixic acid are Firmicutes and Bacteroidetes, while those inhibited by tetracycline are Firmicutes, Bacteroidetes and Cyanobacteria. According to the PICRUSt prediction of metagenome, influence of antibiotics on overall metabolic function of the bacterial community is rather limited. This study has provided valuable information, from a phylogenetic viewpoint, about the influence of high concentration of antibiotics on soil bacterial community.

Structure and dynamics of inactive and active MARK4: conformational switching through the activation process

Microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine protein kinase belonging to a highly-conserved group of PAR proteins that phosphorylate microtubule-associated proteins (tau, MAP2 and MAP4) and regulate cell polarity. MARK4 isoform is mainly found in brain tissue, causing microtubule destabilization in neuronal cells and also tau-protein phosphorylation seen in Alzheimer’s disease. In this study, the dynamic behavior of inactive and active structures of human MARK4 was studied by modeling and molecular dynamics simulations and motions of the protein through activation process were interpolated. Structure and dynamics of the protein active state in presence of Mg-ATP were also studied. The results suggest for occlusion of ATP binding site by activation loop, as the main inactivation mechanism. Data also justify the necessity of UBA (ubiquitin-associated) domain auto-inhibitory role. Within the inactive and active state, G-loop is highly fluctuating and assumes an open conformation and away from Mg-ATP complex. This behavior raises the assumption that this loop may experience other stabilizing interactions with residues out of the kinase core which help its stabilization during the phospho-transfer reaction. Mg-ATP complex localization is well preserved within the catalytic cleft through the electrostatic interactions. However, minor rearrangements of water molecules around this complex are allowed, which may further refine the delicate position of this complex for phospho-transfer reaction.Communicated by Ramaswamy H. Sarma

In vitro Effects of a Novel Silver-based Complex on Influenza Virus

Objective: To study the mechanism of antiviral activity of a new silver-based compound of general formula C6H19Ag2N4 LiO6S2 on influenza virus. Materials and Methods: Antiviral properties were investigated on test culture of Madin–Darby canine kidney (MDCK) cells with serial tenfold dilutions of the influenza virus. The completeness of viral inactivation was confirmed by determining the virus titer after a double passage of virus-containing suspension in MDCK cell culture. Reverse transcription polymerase chain reaction analysis of viral RNA, enzyme immunoassay, and transmission electron microscopy for morphological analysis of influenza virus particles were applied for analysis of viral RNA and effect of substance influence. Results: The results indicate a significant change in the antigenic structure of viral proteins of influenza virus by the silver-containing complex. The main inactivation mechanism of influenza virus by the action of the silver-containing complex is associated primarily with the effect on virion protein structure rather than cleavage of the viral RNA. It was revealed that the mechanism for the inactivation of influenza virus is not associated with cleavage of viral RNA corresponding genes M, NS, or nucleoprotein but more probable with changes of the antigen determinants of virion. It was found a significant damaging effect on influenza virus under action of tested substance at concentration higher than 0.4 mM. Conclusion: The novel silver-based complex possesses antiviral properties and could be considered as a prospective antiviral drug.

An integrated electrolysis – electrospray – ionization antimicrobial platform using Engineered Water Nanostructures (EWNS) for food safety applications

Engineered water nanostructures (EWNS) synthesized utilizing electrospray and ionization of water, have been, recently, shown to be an effective, green, antimicrobial platform for surface and air disinfection, where reactive oxygen species (ROS), generated and encapsulated within the particles during synthesis, were found to be the main inactivation mechanism. Herein, the antimicrobial potency of the EWNS was further enhanced by integrating electrolysis, electrospray and ionization of de-ionized water in the EWNS synthesis process. Detailed physicochemical characterization of these enhanced EWNS (eEWNS) was performed using state-of-the-art analytical methods and has shown that, while both size and charge remain similar to the EWNS (mean diameter of 13 nm and charge of 13 electrons), they possess a three times higher ROS content. The increase of the ROS content as a result of the addition of the electrolysis step before electrospray and ionization led to an increased antimicrobial ability as verified by E. coli inactivation studies using stainless steel coupons. It was shown that a 45-min exposure to eEWNS resulted in a 4-log reduction as opposed to a 1.9-log reduction when exposed to EWNS. In addition, the eEWNS were assessed for their potency to inactivate natural microbiota (total viable and yeast and mold counts), as well as, inoculated E. coli on the surface of fresh organic blackberries. The results showed a 97% (1.5-log) inactivation of the total viable count, a 99% (2-log) reduction in the yeast and mold count and a 2.5-log reduction of the inoculated E. coli after 45 min of exposure, without any visual changes to the fruit. This enhanced antimicrobial activity further underpins the EWNS platform as an effective, dry and chemical free approach suitable for a variety of food safety applications and could be ideal for delicate fresh produce that cannot withstand the classical, wet disinfection treatments.

Castles fall from inside: Evidence for dominant internal photo-catalytic mechanisms during treatment of Saccharomyces cerevisiae by photo-Fenton at near-neutral pH

In this work, the antimicrobial effects of the photo-Fenton process on yeast cells were tested (Saccharomyces cerevisiae), an essential eukaryotic unicellular model of living cells. Near-neutral pH was used in all studies, while iron sulfate and iron citrate were evaluated as iron sources during S. cerevisiae photo-Fenton inactivation under simulated solar light (hv/H2O2/Fe). The following indicators were monitored to decrypt the mechanism of yeast inactivation by neutral photo-Fenton process: cell viability by flow cytometry, damage at DNA level, as well as intracellular and extracellular proteins, assessed by gel electrophoresis. A significant loss of cultivability was monitored through the application of all the different photo-Fenton systems, attributed to the oxidative stresses applied. The mechanisms involved were the homogeneous action of dissolved iron and the heterogeneous action mode of iron oxides. The DNA and protein analyses indicated drastic intracellular damages, while external macromolecules (cell wall and membrane proteins) showed limited degradation. This marked internal photocatalytic processes as the main inactivation mechanism in S. cerevisiae. Different pathways are proposed, forming a general process of inactivation by the near-neutral photo-Fenton systems.

Procesamiento no térmico de alimentos

Conventional approaches to process foods have proven to offer very safe products but, in some cases, the quality of the final product is significantly lower to the original one. Nonthermal processing of foods has emerged as a viable alternative to those conventional processing by offering safe products of excellent quality and at very reasonable cost. These emerging technologies utilize nonthermal microbial stress factors as the main inactivation mechanism. In some cases, external sources of heat or self-generated heat are utilized to supplement the main inactivation mechanism. This combination becomes very relevant in the case of the sterilization of low-acid foods by pressure assisted thermal processing (PATP). This article presents some of the most relevant nonthermal technologies where some of them are already in use by the food industry and others will be adopted in the very near future. It is the case; these nonthermal technologies could be used in combination among themselves or with other preservation approaches seeking synergistic effects in order to have shorter processes and very good quality food products.

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer

The low stability of gammaretroviral and lentiviral vectors affects their production, making high quality clinical preparations a difficult goal to achieve. Recently, our laboratory has shown that the main inactivation mechanism for both these vectors is the loss of their capacity to perform reverse transcription. The present study aimed to increase the stability of gammaretroviral and lentiviral at 37 degrees C and at 4 degrees C. Inactivation studies were performed with gammaretroviral and lentiviral vectors at 37 and 4 degrees C, with and without several stabilizing compounds. The residual viral infectivity and reverse transcription capacity of these samples were tested. The results obtained demonstrate that it is possible to increase the stability of reverse transcription and the infectivity stability of purified gammaretroviral vectors by adding recombinant human albumin (rHSA) to the storage buffer, both at 37 degrees C and at 4 degrees C. For lentiviral vectors, it was observed that further protection was needed. This was achieved by adding lipids to the storage buffer, using a mixture of lipoproteins and rHSA. The difference of stabilization between gammaretroviral and lentiviral vectors was validated by performing stabilization tests with vectors possessing different envelope proteins and produced by different cell lines. The presented study reveals that it is possible to increase the half-life of purified gammaretroviral and lentiviral vectors at 37 degrees C and at 4 degrees C, but the two vectors have different stabilization requirements: for retroviral vectors, the addition of rHSA is enough and, for lentiviral vectors, it is necessary to add both lipoproteins and rHSA. The increase of the stability of the reverse transcription process was shown to have a high impact with respect to the increase of the stability of infectivity.

Somatic APC Inactivation Mechanisms in Sporadic Colorectal Cancer Cases in Hungary

The role of germline inactivation of the adenomatosis polyposis coli (APC) gene in hereditary colorectal cancer is well known, being the most important cause of familial adenomatosus polyposis (FAP) syndrome. Hereditary cases with germline mutations, however, account only for 5-10% of colorectal cancers. The somatic inactivation of this gene has also been observed in sporadic cases. In order to examine the inactivation mechanisms of the APC gene we screened 70 sporadic colorectal cancer cases (27 rectal, 43 intestinal) of different stages for promoter hypermethylation, allelic imbalance (AI) and somatic mutations. The presence of promoter hypermethylation was observed in 21 cases (30%). Fifteen of the examined tumors (21%) showed AI, and also 15 tumors (21%) carried at least one somatic mutation. Thirteen of the detected alterations were novel variations: seven frameshifts, four missense mutations and two polymorphisms. Biallelic inactivation was found in 15 patients (21%). These results suggest that the inactivation of the APC gene is very common in sporadic colorectal cancer, and the main inactivation mechanism of the APC gene is promoter hypermethylation. Allelic imbalance has the same frequency as mutations, and mutations in the APC gene are more common in the early stages and in tumors located in the rectum.

Genetic alterations of the HCCS1 gene in Korean hepatocellular carcinoma.

We analyzed the gene mutations and loss of heterozygosity (LOH) of the HCCS1 gene using intragenic polymorphic markers in a series of 88 primary HCCs. We found two sequence variations at exon 5 and 14 in both normal and tumor DNAs of case 50 and 51, respectively. The variation in case 50 led to a reading frameshift and a premature stop (TGA) at codon 125 and case 51 showed amino acid change at codon 448 (Val-->Ala, GTG-->GCG). Interestingly, these variations were not found in peripheral lymphocytes of 69 normal individuals and 227 cancer patients (86 HCC, 75 unselected gastric cancer, and 66 breast cancer), suggesting that these two variations are mutation, not polymorphism. In addition, we found 14 novel intragenic polymorphic sites in the HCCS1 gene. Thirty-two (47%) of sixty-eight informative cases showed allelic loss at at least one or more intragenic polymorphic sites, but there was no significant relationship between the frequency of LOH and clinicopathologic parameters. These results suggest that mutation of the HCCS1 gene might not be a main inactivation mechanism in the development of Korean HCC and that the HCCS1 gene might be involved in acceleration of the tumorigenic process in Korean HCC.

The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Uptake and metabolism of noradrenaline by the mesenteric arteries of the dog.

Uptake and metabolism of tritiated noradrenaline by the mesenteric arteries of the dog and concomitant behaviour changes in endogenous noradrenaline are described. Posterior mesenteric artery strips were incubated for 30 min in Krebs solution containing (-)-7-3H-noradrenaline (1,084 muM); suitable pretreatments and/or preincubations were performed to study the influence of some drugs on these parameters. The atrial strips showed a high capacity to retain, accumulate and metabolize exogenous noradrenaline. At the concentration of noradrenaline studied, neuronal uptake and deamination represented the main inactivation mechanism, however, when neuronal uptake was blocked, extraneuronal deamination and o-methylation played a more important role, o-Methylation represented a vicarious enzymatic route when monoamine oxidase activity was inhibited. Iproniazid pretreatment increased markedly the endogenous noradrenaline content of the mesenteric arteries. During incubation with tritiated noradrenaline both an outflow of endogenous noradrenaline and an inflow of tritiated noradrenaline were observed.