High level expression,purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Botulinum neurotoxin type E heavy chain consists of two domains: N-terminal half as a translocation domain and C-terminal half (Hcc) as a binding domain. In this research a synthetic gene fragment encoding the binding domain of botulinum neurotoxin type E (BoNT/E-Hcc) was highly expressed in Escherichia coli by pGEX4T-1 vector. After purification, the recombinant BoNT/E-Hcc was evaluated by SDS-PAGE and western blot (immunoblot) analysis. Average yields obtained in this research were 3.7 mg recombinant BoNT/E-Hcc per liter of bacterial culture. The recombinant protein was injected in mice for study of its protection ability against botulinum neurotoxin type E challenges. The challenge studies showed that, vaccinated mice were fully protected against 10⁴ × minimum lethal dose of botulinum neurotoxin type E.     

Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to Hb A1c

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Sedimentary facies and paleoenvironmental interpretation of the Oligocene larger-benthic-foraminifera-dominated Qom Formation in the northeastern margin of the Tethyan Seaway

The well-exposed outcrops of the Bujan, northern Abadeh, and Varkan stratigraphic sections of the Qom Formation in the Iranian part of the “northeastern margin” of the Tethyan Seaway were characterized by abundant biogenic components dominated by foraminifers, coralline red algae, and corals. The Qom Formation is Rupelian–Chattian in age in the study areas. Based on the field investigations, depositional textures, and dominant biogenic components, fifteen (carbonate and terrigenous) facies were identified. These facies can be grouped into four depositional environments: open marine, open lagoon, restricted lagoon, and continental braided streams. The marine facies were deposited on a ramp-type platform. The euphotic inner ramp was characterized mainly by imperforate foraminifera, with co-occurrence of some perforate taxa. These facies passed basinward into a mesophotic (middle) ramp with Neorotalia packstone (F5), coral, coralline algae, perforate foraminiferal packstone (F4), and coral patch reefs (F7). The deeper, oligophotic ramp facies were marly packstones with planktonic and hyaline benthic foraminifera, including large lepidocyclinids and nummulitids. The abundance of perforate foraminifera and the absence of facies indicating restricted lagoonal or intertidal settings suggest that the Varkan section was deposited mainly in open marine settings with normal salinity. The prevalence of larger benthic foraminiferal and red algal assemblages, together with the coral facies, indicates that carbonate production took place in tropical–subtropical waters.     

Tolerance Responses of Two Cotton Cultivars Exposed to Ultraviolet-A: Photosynthetic Performance and Some Chemical Constituents

Stress tolerance of two Egyptian cotton cultivars (Gossypium barbadense L.) (Giza 45 and Giza 86) exposed to various doses (40, 80, 160, and 320 min) of artificial ultraviolet-A (UV-A) radiation (366 nm) was investigated. Seed germination of cv. Giza 86 was promoted at 40 min, progressively inhibited at 80 and 160 min, and completely suppressed at 320 min irradiation. However, seed germination of cv. Giza 45 was not promoted but inhibited by UV-A light and stopped at the dose of 160 min. In contrast to seed germination, seedling growth of cv. Giza 86 was negatively stressed even at 40 min-dose. UV-A radiation reduced leaf carbohydrate content and shoot growth of both cultivars, but the response was comparatively higher in cv. Giza 45. UV-A radiation decreased chlorophyll (Chl) and carotenoid contents in parallel with an increase in the Chl a/b ratio, diminished Hill reaction activity, and quenched Chl a fluorescence independent of the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting an inhibitory effect on both the water-splitting system and electron transport from the primary to the secondary acceptors of photosystem II (PSII) (acceptor side). UV-A radiation also decreased the ratio of unsaturated to saturated fatty acids in thylakoid membranes, thus indicating that the inhibition of PSII activity was followed by lipid peroxidation and changes in thylakoid membrane fluidity. These changes reflect the disturbance of structure, composition, and functioning of the photosynthetic apparatus, as well as the sensitivity of PSII to UV-A stress. Both cultivars developed adaptive mechanisms for damage alleviation involving the accumulation of flavonoids, total lipids, and total soluble proteins, as well as development of smaller and thicker leaf blades. Since cv. Giza 86 showed comparatively higher level of adaptation, it tolerates UV-A stress more successful than cv. Giza 45.     

Chemical Structure and Morphology of Dorsal Root Ganglion Neurons from Naive and Inflamed Mice

Fourier transform infrared spectromicroscopy provides label-free imaging to detect the spatial distribution of the characteristic functional groups in proteins, lipids, phosphates, and carbohydrates simultaneously in individual DRG neurons. We have identified ring-shaped distributions of lipid and/or carbohydrate enrichment in subpopulations of neurons which has never before been reported. These distributions are ring-shaped within the cytoplasm and are likely representative of the endoplasmic reticulum. The prevalence of chemical ring subtypes differs between large- and small-diameter neurons. Peripheral inflammation increased the relative lipid content specifically in small-diameter neurons, many of which are nociceptive. Because many small-diameter neurons express an ion channel involved in inflammatory pain, transient receptor potential ankyrin 1 (TRPA1), we asked whether this increase in lipid content occurs in TRPA1-deficient (knock-out) neurons. No statistically significant change in lipid content occurred in TRPA1-deficient neurons, indicating that the inflammation-mediated increase in lipid content is largely dependent on TRPA1. Because TRPA1 is known to mediate mechanical and cold sensitization that accompanies peripheral inflammation, our findings may have important implications for a potential role of lipids in inflammatory pain.     

C-Phycocyanin Confers Protection against Oxalate-Mediated Oxidative Stress and Mitochondrial Dysfunctions in MDCK Cells

Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.     

PIA and rSesC Mixture Arisen Antibodies Could Inhibit the Biofilm-Formation in Staphylococcus aureus

Background:Staphylococcus aureus as a causative agent of hospital-acquired infections has been considered as the primary concern in biomaterial-related infections (BAIs).Methods:Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface-exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm-forming S. aureus strains was assessed by the semi-quantitative micro-plate procedure.Results:Data showed that the total IgGs were boosted in mice immunized sera. By performing an inhibition assay, the biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing individual antigens in a biofilm inhibition assay.Conclusion:immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus. Hopefully, this study corresponds to the suggestion that the immunization of mice with PIA and rSesC candidate vaccines could protect against S. aureus infection.Key Words: PIA, Purification, Staphylococcus aureus, rSesC, Vaccine candidates     

Isolation and identification of Amycolatopsis sp. strain 1119 with potential to improve cucumber fruit yield and induce plant defense responses in commercial greenhouse

Plant and Soil - The aims of this work were (i) to find a soil indicator to predict durum wheat yield response to Zn fertilization, (ii) to compare the effect of various Zn fertilization strategies...