Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Proline-Functionalized Graphene Oxide Nanoparticles (GO–Pro NPs) Mitigate Salt-Induced Adverse Effects on Morpho-Physiological Traits and Essential Oils Constituents in Moldavian Balm (Dracocephalum moldavica L.)

Many methodologies have been established to lessen negative impacts of salinity on plants. Of those methodologies, nanoparticles (NPs) application has achieved great importance thanks to their unique physico-chemical properties. Consequently, formerly respecting encouraging impacts of graphene oxide (GO) and proline (Pro) on different plant processes under non-stress and stress conditions, proline-functionalized graphene oxide nanoparticles “GO–Pro NPs” were synthesized and characterized. Graphite powder, as starting material, was used to synthesize GO using modified Hummers method followed by functionalization of its surface by proline in basic media. Afterward, GO–Pro NPs, GO and Pro, each at 0, 50 and 100 mg L^?1 concentrations with three replications, were applied on Moldavian balm (Dracocephalum moldavica L.) plants to assay their effects under non-stress (0 mM) and salt stress (50 and 100 mM) conditions. GO–Pro NPs and Pro effectively alleviated negative effects of salinity through increasing morphological parameters, photosynthetic pigments, chlorophyll fluorescence parameters, chlorophyll index (SPAD), and membrane stability index (MSI) and decreasing hydrogen peroxide and malondialdehyde, as well. Also application of GO–Pro NPs enhanced proline, antioxidant enzymes activities, and most dominant constituents of essential oil. The highest MSI (48.87%) and proline content (15.36 µM g^?1 FW) were observed in plant treated with GO–Pro NPs (50 mg L^?1) under 100 mM NaCl salinity stress. The GO–Pro NPs treatment at lower dose (50 mg L^?1) could be introduced as the best preservative treatment for Moldavian balm under salt stress. GO application mostly had no effect on the measured parameters announcing it as carrier for Pro to enhance its efficiency. In conclusion, GO–Pro NPs application could promote Moldavian balm performance and essential oil under salinity presenting GO–Pro NPs as new treatment against stress conditions.

     

Fast killing kinetics,significant therapeutic index,and high stability of melittin-derived antimicrobial peptide

The emergence of multidrug-resistant (MDR) bacteria is a major challenge for antimicrobial chemotherapy. Concerning this issue, antimicrobial peptides (AMPs) have been presented as novel promising antibiotics. Our previous de novo designed melittin-derived peptides (MDP1 and MDP2) indicated their potential as peptide drug leads. Accordingly, this study was aimed to evaluate the kinetics of activity, toxicity, and stability of MDP1 and MDP2 as well as determination of their structures. The killing kinetics of MDP1 and MDP2 demonstrate that all bacterial strains were rapidly killed. MDP1 and MDP2 were ca. 100- and 26.6-fold less hemolytic than melittin and found to be respectively 72.9- and 41.6-fold less cytotoxic than melittin on the HEK293 cell line. MDP1 and MDP2 showed 252- and 132-fold improvement in their therapeutic index in comparison to melittin. MDP1 and MDP2 sustained their activities in the presence of human plasma and were found to be ca. four to eightfold more stable than melittin. Spectropolarimetry analysis of MDP1 and MDP2 indicates that the peptides adopt an alpha-helical structure predominantly. According to the fast killing kinetics, significant therapeutic index, and high stability of MDP1, it could be considered as a drug lead in a mouse model of septicemia infections.

     

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Cowpea Bacterial Wilt in Iran

During surveys in cowpea fields of Marand County, East Azerbaijan province, Iran, in the summer of 2013, a suspected bacterial disease was observed on cowpea leaves as tan spots and interveinal necrotic lesions surrounded by chlorotic margins. The disease was of high incidence where some fields had been fully destroyed and severity of the disease in some fields had reached up to 70%. Gram‐positive, yellow‐pigmented, coryneform bacteria were isolated from infected leaves. Pathogenicity of isolates was confirmed on 20‐day‐old cowpea (cv. Khoy) plants, and they were identified as Curtobacterium flaccumfaciens pv. flaccumfaciens based on biochemical test results confirmed using specific PCR primers. This is the first report of C. flaccumfaciens pv. flaccumfaciens, the causal agent of cowpea bacterial wilt in Iran.     

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome: accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of: cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as: rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G0/G1, S and G2/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

Application of water super absorbents in waste air biofiltration

Water super absorbents are low cross-linked hydrophilic polymers that absorb water in amounts up to several hundred times their dry weight. In this study, the effect of adding these materials to the bed of a biofilter was investigated. Two equal size biofilters were used for this purpose. One of the biofilters was packed with a mixture of perlite and a commercial polyacrylamide based super absorbent (2.3% dry weight), and the other was packed with perlite to perform as a control. The biofilters were inoculated with a bacterial culture that was able to grow on n-hexane as the sole source of carbon and energy. Both biofilters removed up to 90% of the entering pollutants when using an inlet n-hexane concentration of 1 g/m³, and an air flow rate of 0.3 L/min (mass loading of 18.34 g/m³/h, and empty bed residence time of 3.27 min). The super absorbent had a positive effect on the performance of the biofilter. While the difference in the performance of the biofilters was marginal when frequent moistening was applied, the difference was considerable when moistening was less frequent.     

Thermal imaging of the periorbital regions during the presentation of an auditory startle stimulus

Infrared thermal imaging of the inner canthi of the periorbital regions of the face can potentially serve as an input signal modality for an alternative access system for individuals with conditions that preclude speech or voluntary movement, such as total locked-in syndrome. However, it is unknown if the temperature of these regions is affected by the human startle response, as changes in the facial temperature of the periorbital regions manifested during the startle response could generate false positives in a thermography-based access system. This study presents an examination of the temperature characteristics of the periorbital regions of 11 able-bodied adult participants before and after a 102 dB auditory startle stimulus. The results indicate that the startle response has no substantial effect on the mean temperature of the periorbital regions. This indicates that thermography-based access solutions would be insensitive to startle reactions in their user, an important advantage over other modalities being considered in the context of access solutions for individuals with a severe motor disability.     

Novel recombination system using Cre recombinase alpha complementation

A major limitation for the use of Cre recombinase is its toxicity and a lack of temporal control over its activity. We have developed a new recombination system using Cre recombinase α-complementation. Cre recombinase was divided and one fragment (β) was introduced into cells between two loxP sites with a CMV promoter in the upstream. The gene of interest (EGFP) was positioned just downstream of this construct. Cre recombinase activity was recovered by adding the other part of the molecule (α) to cells as a protein fragment, as evidenced by the expression of EGFP under the control of the CMV promoter. The activity of fragmented cre reached 68% of that of the wild type enzyme at 1 μM α-protein.