Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems

Background: Phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH. Methods: The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.     

Microalgae as Contributors to Produce Biopolymers

Biopolymers are very favorable materials produced by living organisms, with interesting properties such as biodegradability, renewability, and biocompatibility. Biopolymers have been recently considered to compete with fossil-based polymeric materials, which rase several environmental concerns. Biobased plastics are receiving growing interest for many applications including electronics, medical devices, food packaging, and energy. Biopolymers can be produced from biological sources such as plants, animals, agricultural wastes, and microbes. Studies suggest that microalgae and cyanobacteria are two of the promising sources of polyhydroxyalkanoates (PHAs), cellulose, carbohydrates (particularly starch), and proteins, as the major components of microalgae (and of certain cyanobacteria) for producing bioplastics. This review aims to summarize the potential of microalgal PHAs, polysaccharides, and proteins for bioplastic production. The findings of this review give insight into current knowledge and future direction in microalgal-based bioplastic production considering a circular economy approach. The current review is divided into three main topics, namely (i) the analysis of the main types and properties of bioplastic monomers, blends, and composites; (ii) the cultivation process to optimize the microalgae growth and accumulation of important biobased compounds to produce bioplastics; and (iii) a critical analysis of the future perspectives on the field.     

Transparent Polycaprolactam Electrospun Nanofibers Doped with 1,10-phenanthroline Optical Sensor for Colorimetric Determination of Iron (II) and Vitamin C

A novel optical chemical sensor based on a transparent electrospun nanofibrous scaffold, composed of polycaprolactam (PA6) and 1,10-phenanthroline (Phen), deposited on a glass slide and impregnated with polyvinyl alcohol (PVA) was coupled with UV-vis spectrophotometry and used for colorimetric determination of ferrous ion (Fe²⁺) and ascorbic acid (AA). The electrospun nanofibers were characterized using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) techniques. The main factors affecting performance of the optical sensor (PA6-Phen/PVA@glass) including Phen dosage, pH of sample solution, electrospinning time, polymer solution flowrate, electrospinning voltage, and PVA amount were investigated and the related optimum values were obtained. The analytical merits of the sensor for quantitative determination of Fe²⁺ and ascorbic acid was evaluated. The limit of detection (LOD) and limit of quantification (LOQ) for Fe²⁺ were 1 and 3 μg mL^-1, respectively. The linear dynamic range (LDR) was 3–150 μg mL^-1 with the determination coefficient (R²) of 0.991. The relative standard deviation (RSD %, n=3) for a solution of 60 μg mL^-1 was 5.4 %. For determination of AA, LOD and LOQ were obtained equal to 0.5 and 2 μg mL^-1, respectively. The linear dynamic range was in the 2–200 μg mL^-1 range with a R2 of 0.994. The RSD % at 100 μg mL^-1, n=3) was equal to 7.0 %. The sensor was applied successfully to the detection of Fe(II) and AA in real water samples and aspirin tablets.     

Effect of oleuropein on tissue myeloperoxidase activity in experimental spinal cord trauma

Background: Neutrophil infiltration plays an important role in inflammatory reactions following spinal cord injury (SCI) and these cells cause substantial secondary tissue damage. The purpose of this study was to determine the effect of oleuropein (OE) on myeloperoxidase (MPO) activity as an index of neutrophil infiltration. Methods: Rats were randomly divided into four groups of 7 rats each as follows: sham-operated group, trauma group, and OE treatment groups (20 mg/kg, i.p., immediately and 1 hour after SCI). Spinal cord samples were taken 24 hours after injury and studied for determination of MPO activity. Results: The results showed that MPO activity was significantly decreased in OE-treated rats. Conclusion: On the basis of our findings, we propose that OE may be effective in protecting rat spinal cord from secondary damage by modulating of neutrophil infiltration.Key Words: Oleuropein (OE), Neutrophil infiltration, Myeloperoxidase     

Determination of fiber length and juvenile and mature wood zones in Acer velutinum Boiss. trees grown in Iran

In our investigation we studied fiber lengths and the transition age from juvenile to mature wood in Acer velutinum Boiss. For this purpose, samples from three normal maple trees at a Noshahr site in northern Iran were selected. Disks were cut at breast height. Test samples were taken along a radial direction from the pith to the bark, accounting for every ring during a 48-year period. We used the Franklin method to distinguish between fibers of juvenile and mature wood. The results show that the fiber length increased along the radial direction from the pith to the bark. The transition age between juvenile and mature wood was determined at the 14th annual ring from the pith.     

Anti-inflammatory activities of cucurbitacin E isolated from Citrullus lanatus var. citroides: role of reactive nitrogen species and cyclooxygenase enzyme inhibition

The in vivo and in vitro mechanistic anti-inflammatory actions of cucurbitacin E (CE) (Citrullus lanatus var. citroides) were examined. The results showed that LPS/INF-γ increased NO production in RAW264.7 macrophages, whereas L-NAME and CE curtailed it. CE did not reveal any cytotoxicity on RAW264.7 and WRL-68 cells. CE inhibited both COX enzymes with more selectivity toward COX-2. Intraperitoneal injection of CE significantly suppressed carrageenan-induced rat's paw edema. ORAC and FRAP assays showed that CE is not a potent ROS scavenger. It could be concluded that CE is potentially useful in treating inflammation through the inhibition of COX and RNS but not ROS.     

Effects of nano-silver impregnation on brittleness, physical and mechanical properties of heat-treated hardwoods

The present study investigates the effect of heat treatment of untreated and nano-silver-impregnated Populus nigra, Populus deltoides and Fagus orientalis on the physical and mechanical properties. Specimens were impregnated with a 200-ppm aqueous silver nanoparticles suspension. Heat treatment was carried out at temperatures of 135 ± 3 °C and 185 ± 3 °C. Nano-silver impregnation aggravated the effects of heat treatment. Although significant in some cases, nano-silver impregnation did not seem to have great effect on physical properties. It may be concluded that although NS impregnation aggravated the effects of heat treatment on solid woods, other factors may also be involved such as the species and density of the wood.     

Effects of emulsified versus saline administration of GnRHa on induction of ovulation in rainbow trout, Oncorhynchus mykiss

The effects of saline-dissolved or Freund's incomplete adjuvant (FIA)-emulsified GnRHa treatment on the induction of ovulation in rainbow trout, Oncorhynchus mykiss, were examined. The FIA-emulsified GnRHa was first diluted in 0.25 ml physiological saline and then mixed with an equal volume of FIA. Fish were selected in the beginning of the spawning season and were allocated into four groups and were treated intraperitoneally with (a) 0.5 ml of emulsified GnRHa (GnRHa–FIA), (b) 0.5 ml saline-dissolved GnRHa in a single injection (GnRHa-1), (c) 0.5 ml saline-dissolved GnRHa in two injections spaced 1 d apart (GnRHa-2) and (d) 0.5 ml of saline (Control). The GnRHa dose in all hormone treatments was 25 µg kg^− 1. All fish in the FIA–GnRHa and GnRHa-2 groups ovulated within 10 and 11 d after treatment, respectively. In contrast, only 75% in the Control fish and 60% of the fish in the GnRHa-1 group ovulated within 36 d after treatment. None of the treatments caused any pre- or post-spawning mortality in the broodstock. Fertilization, eyeing and hatching percentages of the produced progeny were normal in all the treatment groups and did not differ significantly among them. In conclusion, FIA-emulsified GnRHa can be effective in advancing the onset of and synchronizing the ovulation of rainbow trout within a two-week period, thus shortening the egg collection period, without affecting broodstock survival and egg quality.     

Microsatellite and mitochondrial DNA analyses reveal no genetic difference between two pufferfish species torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and karasu <Emphasis Type="Italic">T. chinensis</Emphasis>

It has been reported that the nucleotide sequences of the mitochondrial and nuclear genes of Takifugu pufferfish torafugu T. rubripes and karasu T. chinensis show 99–100% sequence identity, indicating a very close relationship between the two species. To further investigate this genetic relationship, we compared genetic variation at four microsatellite loci and at the mitochondrial control region (CR) (561 bp) between groups of T. rubripes caught at two locations [TrG, caught in the Genkai Sea off Tsushima Island in 2003 (n = 50); TrS, caught in the Suwo Sea off Kita-Kyushu in 2008 (n = 50)] and T. chinensis caught at one location (TcK, caught off the east coast of Korea in 2004; n = 50). Analyses using microsatellite loci showed that genetic diversity index values of the TrG, TrS and TcK groups were 0.9505, 0.9350 and 0.9335, respectively, while values of genetic distance and genetic differentiation between TrG and TcK (0.0543 and 0.0189, respectively) were smaller than those between TrG and TrS (0.0857 and 0.0194, respectively). Analyses using CR for the same specimens showed that genetic distances were consistent with those obtained using microsatellite loci. These results, together with our previous observations, suggest that T. rubripes and T. chinensis are very closely related and possibly can be regarded as the same species.