Ethidium bromide: destruction and decontamination of solutions

Ethidium bromide in water, TBE buffer, Mops buffer, and cesium chloride solution may be completely degraded by reaction with sodium nitrite and hypophosphorous acid. Only non-mutagenic reaction mixtures were produced. Destruction was greater than 99.8% in all cases; the limit of detection was 0.5 micrograms ethidium bromide per milliliter of solution. Ethidium bromide also may be removed completely from the above solutions by using Amberlite XAD-16 resin. The limit of detection was 0.05 micrograms ethidium bromide per milliliter of solution (0.27 micrograms/ml when cesium chloride solution was used).     

Antimicrobial blue light for decontamination of platelets during storage

Platelet (PLT) storage is currently limited to 5 days in clinics in the United States, in part, due to an increasing risk for microbial contamination over time. In light of well‐documented antimicrobial activity of blue light (405‐470 nm), we investigated potentials to decontaminate microbes during PLT storage by antimicrobial blue light (aBL). We found that PLTs produced no detectable levels of porphyrins or their derivatives, the chromophores that specifically absorb blue light, in marked contrast to microbes that generated porphyrins abundantly. The difference formed a basis with which aBL selectively inactivated contaminated microbes prior to and during the storage, without incurring any harm to PLTs. In accordance with this, when contamination with representative microbes was simulated in PLT concentrates supplemented with 65% of PLT additive solution in a standard storage bag, all “contaminated” microbes tested were completely inactivated after exposure of the bag to 405 nm aBL at 75 J/cm² only once. While killing microbes efficiently, this dose of aBL irradiation exerted no adverse effects on the viability, activation or aggregation of PLTs ex vivo and could be used repeatedly during PLT storage. PLT survival in vivo was also unaltered by aBL irradiation after infusion of aBL‐irradiated mouse PLTs into mice. The study provides proof‐of‐concept evidence for a potential of aBL to decontaminate PLTs during storage.      

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.     

Antimicrobial action and efficiency of silver-loaded zeolite X

Aims: To synthesize silver-loaded zeolite X and establish the extent to which it persist in its antimicrobial action against strains of Escherichia coli K12W-T, Pseudomonas aeruginosa NCIMB8295 and Staphylococcus aureus NCIMB6571. Methods and Results: The antimicrobial action and efficacy of silver-loaded zeolite X on Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were investigated. Zeolite X was synthesized and loaded with Ag⁺ by ion exchange. This resulted in 2·0% (w/w) loading of Ag⁺ in the zeolite framework and 5·8% (w/w) on the zeolite. Escherichia coli and Pseudomonas aeruginosa and Staphylococcus aureus suspended in tryptone soya broth were exposed to 0·15, 0·25, 0·5 or 1·0 g l⁻¹ of silver-loaded zeolite X for a period up to 24 h. No viable cells were detected for any of the three micro-organisms within 1 h. Silver-loaded zeolite X, retrieved three times from the first exposure cultures, was washed with de-ionized water and added to fresh bacterial suspensions. The results showed that the silver-loaded zeolite X retained its antimicrobial action. Conclusions: Silver-loaded zeolite X persisted in its antimicrobial action against all three micro-organisms. Significance and Impact of the study: The results are significant for the longevity of antimicrobial action of silver-loaded zeolite X.     

Antimicrobial peptides with stability toward tryptic degradation

The inherent instability of peptides toward metabolic degradation is an obstacle on the way toward bringing potential peptide drugs onto the market. Truncation can be one way to increase the proteolytic stability of peptides, and in the present study the susceptibility against trypsin, which is one of the major proteolytic enzymes in the gastrointestinal tract, was investigated for several short and diverse libraries of promising cationic antimicrobial tripeptides. Quite surprisingly, trypsin was able to cleave very small cationic antimicrobial peptides at a substantial rate. Isothermal titration calorimetry studies revealed stoichiometric interactions between selected peptides and trypsin, with dissociation constants ranging from 1 to 20 microM. Introduction of hydrophobic C-terminal amide modifications and likewise bulky synthetic side chains on the central amino acid offered an effective way to increased half-life in our assays. Analysis of the degradation products revealed that the location of cleavage changed when different end-capping strategies were employed to increase the stability and the antimicrobial potency. This suggests that trypsin prefers a bulky hydrophobic element in S1' in addition to a positively charged side chain in S1 and that this binding dictates the mode of cleavage for these substrates. Molecular modeling studies supported this hypothesis, and it is shown that small alterations of the tripeptide result in two very different modes of trypsin binding and degradation. The data presented allows for the design of stable cationic antibacterial peptides and/or peptidomimetics based on several novel design principles.     

Antimicrobial efficiency of titanium dioxide‐coated surfaces

Aims: Development and evaluation of an antimicrobially active titanium dioxide coating. Methods and results: For this purpose, titanium dioxide coatings were applied to glass slides by using a sol‐gel method and then exposed to a light source. The antimicrobial efficiency was determined by a count reduction test for selected test strains (Aspergillus niger, Bacillus atrophaeus, Kocuria rhizophila), which were homogenously sprayed onto surface. The bacterial count of K. rhizophila was reduced by up to 3·3 log₁₀ on titanium dioxide samples within 4 h of UV‐A light exposure. Experiments with spore formers did not lead to any significant log reduction. A further aspect of this work was to evaluate the effect of selected parameters (relative humidity, inoculation density, radiation intensity) on the antimicrobial efficiency to gain knowledge for further optimization procedures. At a high relative humidity (85% r.h.), increased inactivation was observed for K. rhizophila (up to 5·2 log₁₀). Furthermore, a dependency of the antimicrobial effect on the radiation intensity and the inoculation density was identified. Conclusions: Antimicrobial surfaces and coatings based on titanium dioxide have the potential to effectively inactivate vegetative micro‐organisms. Significance and impact of the study: Knowledge about the antimicrobial efficiency of titanium dioxide was gained. This is a prerequisite for industrial applications to improve hygiene, food quality and safety.     

The efficiency of strict reverse isolation and antimicrobial decontamination in remission induction therapy of acute leukemia

The efficiency of strict reverse isolation and antimicrobial decontamination in remission induction therapy of acute leukemia was studied retrospectively in 47 patients who were treated with a standardized aggressive chemotherapy of daunorubicin and cytosine arabinoside. Twenty-two patients were treated in strict reverse isolation with antimicrobial decontamination and 25 patients in the open ward without any measures against infections. In the patients in isolation the incidence of new infections per patient was 0.77 compared to 1.42 in the control group. The rate of complete remissions was 77% in the patients in isolation vs. 56% in the control patients.     

Solubility and stability of sterigmatocystin in aqueous solutions

Mycotoxin Research - In the present work we studied the ability of phosphate buffer to solubize sterigmatocystin (ST) at different pH values. We observed a higher solubility of ST at acid pH...     

Relationship between conformational stability and amplification efficiency of prions

Recent studies demonstrated that the efficiency, rate, and yield of prion amplification in vitro could be substantially improved by supplementing protein misfolding cyclic amplification (PMCA) with Teflon beads [Gonzalez-Montalban et al. (2011) PLoS Pathog. 7, e1001277]. Here we employed the new PMCA format with beads (PMCAb) to gain insight into the mechanism of prion amplification. Using a panel of six hamster prion strains, the effect of beads on amplification was found to be strain-specific, with the largest improvements in efficiency observed for strains with the highest conformational stability. This result suggests a link between PrP(Sc) conformational stability and its fragmentation rate and that beads improved amplification by assisting fragmentation. Furthermore, while exploring the PrP(Sc)-independent bead effect mechanism, a synergy between the effects of RNA and beads on amplification was observed. Consistent with previous studies, amplification of all six hamster strains tested here was found to be RNA-dependent. Under sonication conditions used for PMCA, large RNA molecules were found to degrade into smaller fragments of a size that was previously shown to be the most effective in facilitating prion conversion. We speculate that sonication-induced changes in RNA size distribution could be one of the rate-limiting steps in prion amplification.     

Synthesis and stability of two indomethacin prodrugs

The purpose of this study was to synthesize and study the in vitro enzymatic and non-enzymatic hydrolysis of indomethacin-TEG ester and amide prodrugs. It was found that the ester conjugate 10 was comparatively stable between pH 3 and 6 (half-life>90h), with a half-life equal to 5.2h in 80% buffered plasma. In contrast, the amide conjugate 12 appeared to be stable over the entire pH range studied with the only observed degradation being cleavage of the indolic N-4-chlorobenzoyl moiety.     

Thermal stability of aminoacyl-tRNAs in aqueous solutions

Life in hot environments poses certain constraints on the metabolism of thermophilic organisms. Many universal metabolic intermediates are quite labile compounds, and without protection will rapidly decompose at elevated temperatures. Among these are aminoacyl-tRNAs that are necessarily formed upon functioning of the translation apparatus. Aminoacyl-tRNAs are known to be hydrolyzed rapidly even at moderate temperatures under mild alkaline conditions. We studied the thermal stability of phenylalanyl- and alanyl-tRNA in aqueous solutions in order to evaluate a potential threat posed by high temperatures to these components of the translation machinery of thermophiles. Specific second-order rate constants of the aminoacyl-tRNA hydrolysis reaction were determined in the range 20 degrees -80 degrees C. The activation energy of phenylalanyl- and alanyl-tRNA hydrolysis was found to be about 42 and 23 kJ/mol, respectively. The calculated half-lives of aminoacyl-tRNAs at sub-80 degrees C temperatures vary from several seconds to several dozens of seconds at near-neutral pH. The possible mechanisms counteracting the observed thermolability of aminoacyl-tRNAs in vivo are discussed.     

Relative efficiency of abdominal muscles in spine stability

Using an iterative kinematics-driven nonlinear finite element model, relative efficiency of individual abdominal muscles in spinal stability in upright standing posture was investigated. Effect of load height on stability and muscle activities was also computed under different coactivity levels in abdominal muscles. The internal oblique was the most efficient muscle (compared with the external oblique and rectus abdominus) in providing stability while generating smaller spinal loads with lower fatigue rate of muscles. As the weight was held higher, stability deteriorated requiring additional flexor-extensor activities. The stabilising efficacy of abdominal muscles diminished at higher activities. The difference in critical loads in frontal and sagittal planes computed in the absence of abdominal coactivity disappeared under prescribed coactivities suggesting an optimal system in stability. The central nervous system may settle for a less stable spine in favour of lowering the risk of injury. Findings could help introduce stability criterion in optimisation models.     

Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.

The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1.     

Global stability of two species systems

Summary Sufficient conditions for the global stability of equilibria in general, nonlinear, two-species model ecosystems are derived. These results contain and extend the theorem of Goh (1976), which is specific to linear per capita growth rates.     

Antimicrobial activity and stability of protonectin with D‐amino acid substitutions

The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D ‐amino acids. Both the D ‐enantiomer of protonectin (D ‐prt) and D ‐Lys‐protonectin (D ‐Lys‐prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D ‐prt showed strong stability against trypsin, chymotrypsin and the human serum, while D ‐Lys‐prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D ‐Lys‐prt still kept typical α‐helical structure in the membrane mimicking environment, while D ‐prt showed left hand α‐helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D ‐amino acid substitution or partially D ‐amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D ‐prt and D ‐Lys‐prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.