Localized surface plasmon resonance based gold nanobiosensor: Determination of thyroid stimulating hormone

An immunoassay method based on the peak shift of the localized surface plasmon resonance (LSPR) absorption maxima has been developed for the determination of the thyroid stimulating hormone (TSH) in human blood serum. The anti-TSH antibody was adsorbed on the synthesized gold nanoparticles by electrostatic forces. The efficiency of the nanobiosensor was improved by optimizing the factors affecting the probe construction such as the pH and the antibody to gold nanoparticles ratio. Dynamic light scattering was applied for the characterization of the constructed probe. The amount of peak shift of the LSPR absorption maxima was selected as the basis for determination of TSH antigen. The linear dynamic range of 0.4–12.5 mIU L⁻¹ and the calibration sensitivity of 1.71 L mIU⁻¹ were obtained. The human control serum sample was analyzed for TSH by constructed nanobiosensor and the acceptable results were obtained.     

Antixenosis and antibiosis response of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) to two-spotted spider mite (<Emphasis Type="Italic">Tetranychus urticae</Emphasis>)

The two-spotted spider mite, Tetranychus uticae Koch (Acari: Tetranychidae), is globally one of the most devastating pests that feed on numerous crops, including common bean (Phaseolus vulgaris L.). This study was aimed to evaluate the effects of genotype and morphological attributes of common bean on T. uticae. Forty common bean accessions were used to investigate antixenosis and antibiosis through assessing mite feeding preference and reproduction under laboratory conditions. Three resistant (i.e., 56, 63, 238) and two susceptible (i.e., 182, 236) accessions, along with cultivars Naz (resistant) and Akhtar (susceptible), were used in a life-table study. Both antixenosis and antibiosis mechanism were observed in all of the accessions, albeit a negative correlation occurred. Significant differences were observed for all traits of T. urticae: developmental time of immature stages, reproduction, adult longevity and life-table parameters. Based on the intrinsic rate of increase, the accessions 56, 63, 182, 238, and cv. Naz impose high antibiotic effects on T. urticae. Although significant variation existed among accessions for morphological factors, only glandular trichomes correlated with mite fecundity and feeding preference.     

Development of a localized surface plasmon resonance-based gold nanobiosensor for the determination of prolactin hormone in human serum

A localized surface plasmon resonance immunoassay has been developed to determine prolactin hormone in human serum samples. Gold nanoparticles were synthesized, and the probe was prepared by electrostatic adsorption of antibody on the surfaces of gold nanoparticles. The pH and the antibody-to-gold nanoparticle ratio, as the factors affecting the probe functions, were optimized. The constructed nanobiosensor was characterized by dynamic light scattering. The sensor was applied for the determination of prolactin antigen concentration based on the amount of localized surface plasmon resonance peak shift. A linear dynamic range of 1–40 ng ml⁻¹, a detection limit of 0.8 ng ml⁻¹, and sensitivity of 10 pg ml⁻¹ were obtained. Finally, the nanobiosensor was applied for the determination of prolactin in human control serum sample.     

Design and development of a whole-cell luminescent biosensor for detection of early-stage of apoptosis

We present here a novel whole-cell biosensor to detect early-stages of apoptosis based on Apaf-1 oligomerization and apoptosome formation using the split luciferase strategy. The amino-fragment (1-416 amino acids) and carboxy-fragment (395-550 amino acids) of firefly luciferase were fused to amino-terminal of Apaf-1. The cotransfected HEK cells were then treated with doxorubicin for induction of apoptosis. The performance of our biosensor for monitoring of programmed cell death over 24h was investigated by measuring bioluminescence activities. We observed a significant increase (～15 fold) in luminescence signal compared to control cells 4h after apoptosis induction. It reached a maximum activity over 10h (～155 fold). Moreover, juxtapositioning of Apaf-1 monomer and apoptosome formation occur about 5h earlier than the appearance of significant caspase3/7 activity upon induction of apoptosis by doxorubicin. The time-response curve of split luciferase shows a sigmoidal pattern which indicates cooperativity in oligomerization of Apaf-1 upon binding of cytochrome c. This biosensor can be used as a new platform, based on the protein fragment complementation strategy for assessing potential chemotherapeutic drugs as well as a sensitive and dynamic system in the time- and dose-dependent studies of apoptosis.     

In vitro cytotoxicity studies of parent and nanoencapsulated Holmium-2,9-dimethyl-1,10-phenanthroline complex toward fish-salmon DNA-binding properties and antibacterial activity

Abstract

In this study, the interaction of Holmium (Ho) complex including 2, 9-dimethyl-1,10-phenanthroline, also called Neocuproine (Neo), [Ho(Neo)₂Cl₃.H₂O], as fluorescence probe with fish-salmon DNA (FS-DNA) is studied during experimental investigations. Multi-spectroscopic methods are utilized to determine the affinity binding constants (K_b) of complex–FS-DNA. It is found that fluorescence of Ho complex is strongly quenched by the FS-DNA through a static quenching procedure. Under optimal conditions in Tris(trishydroxymethyl-aminomethane)–HCl buffer at 25?°C with pH?≈?7.2, intrinsic binding constant K_b of Ho complex is 6.12?±?0.04?×?10⁵ M^?1. Also, the binding site number and Stern–Volmer quenching constant are calculated. There are different approaches, including iodide quenching assay, salt effect and thermodynamical assessment to determine the features of the binding mode between Ho complex and FS-DNA. Also, the parent and starch and lipid nanoencapsulated Ho complex, as potent antitumor candidates, were synthesized. The main structure of Ho complex is maintained after encapsulation using starch and lipid nanoparticles. 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to assess the anticancer properties of Ho complex and its encapsulated forms on human cancer cell lines of human lung carcinoma cell line and breast cancer cell line. In conclusion, these compounds could be considered as new antitumor candidates.

Communicated by Ramaswamy H. Sarma     

Heterologous Expression,Purification and Characterization of a Peroxidase Isolated from <Emphasis Type="Italic">Lepidium draba</Emphasis>

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.     

Spermine Pre-Treatment Improves Some Physiochemical Parameters and Sodium Transporter Gene Expression of Pumpkin Seedlings under Salt Stress

It is known that polyamines (PAs) including spermine (Spm) enhance the abiotic stress tolerance of crops. Here, the effects of hydroponic Spm pre-treatment on the amelioration of the adverse effects of salinity were investigated in pumpkin (Cucurbita pepo L.) that is an economically important horticultural crop and sensitive to salinity, especially at the establishment stage. For this purpose, 10-day-old, uniform-sized seedlings were transplanted to plastic containers containing Hoagland nutrient solution. Spm was added at 0.1 and 1 mM to the hydroponic medium for 5 days before stress. The plants were treated with 40 and 80 mM NaCl for inducing salinity stress. Salt stress reduced the plant growth and potassium content in roots, and these detrimental effects were alleviated when plants were pre-treated with Spm. Salinity stress caused a significant increase in sodium and γ-amino butyric acid (GABA) content when compared with controls. Spm pre-treatment ameliorated these salinity stress effects by increasing sodium content of root and leaves GABA content. Expression analysis of two sodium transporter genes, salt overly sensitive1 (SOS1) and Na⁺/H⁺ exchanger (NHX1) revealed that their expression was differentially induced in roots of plants treated either with salinity or Spm. These results suggest that Spm via the overexpression of the NHX1 gene substantially increased the tolerance to stress by sequestering excess Na+ into the vacuoles and sustaining a better cellular environment. Moreover, Spm has potential to scavenge directly free radical and to alleviate growth inhibition and promote the activity of antioxidant system enzymes in pumpkin seedlings under salt stress.     

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum–chloride colorimetric and Folin–Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125 ng/mL of DOX and 0.625 mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p?<?0.05) and down-regulation of BCL₂ (p?<?0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

     

Silicon and nitric oxide synergistically modulate the production of essential oil and rosmarinic acid in Salvia officinalis under Cu stress

Protoplasma - The individual impact of silicon (Si) and nitric oxide (NO) on secondary metabolism in several plant species has been reported, but their combined effect has never been evaluated yet....