Label-free high-throughput and real-time detections of protein interactions by oblique-incidence reflectivity difference method

Selected Mouse IgG of 1 mg/mL as target was fabricated on microarray for 500 sample dots.Label-free and real-time reaction dynamic processes were detected between the microarrays with Goat Anti-mouse IgG of 0.02 mg/mL using the obliqueincidence reflectivity difference(OIRD)method.We obtained the reaction results and the reaction dynamic curves of 500 protein dots.In addition,we also used label-free detection of protein microarrays of 10080 sample dots,including BSA and different concentrations of Mouse IgG and Rabbit IgG,by OIRD.The obtained reaction results between the protein microarray with 1 mg/mL Goat Anti-mouse IgG and 1 mg/mL Goat Anti-rabbit IgG are reported herein.Experimental results show that OIRD can be not only label-free high-throughput detection method for biological microarrays but also label-free real-time detection in the interaction processes of biomolecules.     

An Improved Kinetic 5′Nucleotidase Assay

Abstract

5′nucleotidase activity was measured by a coupled optical assay in which adenosine, liberated by action of the primary enzyme, released ammonia which in turn formed L-glutamate from 2-oxoglutarate and NADH. Oxidation of the latter is monitored at 340 nm. Greater activity was obtained when triethanolamine buffer, pH 7.2, and Mn⁺⁺ were substituted for tris buffer, pH 7.9, and Mg⁺⁺. The concentration of substrate, 5′AMP, was altered to 1 mmole/liter and that of β-glycerophosphate to 50 mmoles/liter to achieve optimal activity of true nucleotidase and suppression of 5′AMP-hydrolysis by non-specific phosphatases.     

Detection of DNA Hybridization via Fluorescence Intensity Variations of ZnSe-DNA Quantum Dot Biosensors

ZnSe quantum dots (QDs) that were capped with 11-mercaptoundecanoic acid (MUA) and conjugated to amino-modified ssDNA molecules exhibited variations in fluorescence emission intensity upon hybridization with complementary ssDNA in solution, a phenomenon that can be exploited for rapid detection of free ssDNA sequences. Conjugation of MUA-capped ZnSe QDs to amino-modified ssDNA molecules resulted in increased fluorescence emission intensity and stability at room temperature. Increasing the length of the ssDNA, that was conjugated to the QDs, resulted in increased fluorescence emission intensity up to a length of about 50 nucleotide bases, beyond which the peak emission intensity reached a plateau. Hybridization of QD-ssDNA conjugates with complementary ssDNA, either in free form or bound to QDs from the same population, resulted in additional fluorescence emission intensity amplification. A small red shift was observed when three-dimensional QD-dsDNA-QD structures were formed. The QD-ssDNA sensors with single ssDNA molecule per QD were developed and used for rapid quantitative detection of fully or partially complementary free ssDNA sequences in aqueous solution. Partial hybridization of the QD-ssDNA sensors with short ssDNA targets resulted in smaller QD emission intensity amplification, when compared to full hybridization. A QD-ssDNA sensor containing a sequence corresponding to the hemoglobin beta gene was used to detect and discriminate between free ssDNA targets consisting of a complementary ssDNA sequence and targets containing a single-base mutation that can cause sickle-cell anemia. Such QD-based biosensors can form the basis for rapid separation-free assays that can be used to detect target biomolecules in solution.     

Polyphenols from Citrus Tacle® Extract Endowed with HMGCR Inhibitory Activity: An Antihypercholesterolemia Natural Remedy

Tacle^® is a citrus fruit obtained from the crossbreeding of Clementine and Tarocco cultivars. This fruit retains a promising nutraceutical potential most likely due to a high content in polyphenols, among which the main constituents are the two glycosides naringin and hesperidin. Herein, we evaluated, through an in vitro assay, the capability of Tacle extracts to inhibit the hydroxymethylglutaryl-CoA reductase enzyme, which plays a key role in cholesterol biosynthesis. The results obtained spurred us to investigate whether the anti-enzymatic activity observed may be due to a direct interaction of aglycones naringenin and hesperetin with the enzyme catalytic site. Molecular docking simulations indicated that these two compounds are able to anchor to the protein with binding modes and affinities similar to those found for statins, which represent mainstream medications against hypercholesterolemia. The overall results showed an interesting nutraceutical potential of Tacle, suggesting that its extract could be used for dietary supplementation in the treatment of moderate hypercholesterolemia.     

人肝癌SMMC-7721细胞对低剂量γ射线辐射超敏感性的研究

研究了人肝癌细胞SMMC-7721在低剂量γ射线照射下超敏感性和增强的辐射抗性响应。选用对数生长期细胞接受0—6 Gy不同剂量的^60Coγ射线的照射。利用流式细胞仪对细胞进行分选计数,并用克隆形成法检测细胞存活率。发现SMMC-7721细胞存在低剂量辐射超敏感性和增强的辐射抗性响应,即在0—0．3Gy之间细胞表现出单位剂量杀伤增强现象,在0．3—1Gy细胞表现一定的辐射抗性,在1Gy以上,细胞的存活符合线性平方模型。     

Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity

A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.     

Chemical composition and antioxidant activity of aerial parts of Ferula longipes Coss. ex Bonnier and Maury

This is the first study on the phytochemistry and antioxidant activity of Ferula longipes Coss. ex Bonnier and Maury (Apiaceae). A new flavonoid quercetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-[2-O-caffeoyl]-glucopyranoside (1), along with 10 known compounds kaempferol-3-O-α-L-rhamnopyranoside (2), quercetin-3-O-α-L-rhamnopyranoside (3), kaempferol-3-O-ß-D-glucopyranoside-7-O-α-L-rhamnopyranoside (4), isorhamnetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-glucopyranoside (5), quercetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-glucopyranoside (6), isorhamnetin-3,7-di-O-β-D-glucopyranoside (7), apigenin (8), apigenin-7-O-ß-D-glucopyranoside (9), 3,5-dicaffeoylquinic acid (10), deltoin (11) were isolated from the aerial parts of Ferula longipes Coss. Structures elucidation was performed by comprehensive 1D and 2D NMR analyses, mass spectrometry and by comparison with literature data. The compounds 1, 3, 4, 6, 7 and 10 were evaluated for their antioxidant activity, compound 1 exhibited the best antiradical activity potential and showed IC₅₀ and A_0.5 values 5.70, 7.25, 5.00, and 2.63 μg/mL towards DPPH free radical-scavenging, ABTS, CUPRAC, and reducing power assays, respectively compared with BHA, BHT and ascorbic acid which were used as positive controls.     

Odorant Receptor 7D4 Activation Dynamics

Deciphering how an odorant activates an odorant receptor (OR) and how changes in specific OR residues affect its responsiveness are central to understanding our sense of smell. A joint approach combining site‐directed mutagenesis and functional assays with computational modeling has been used to explore the signaling mechanics of OR7D4. In this OR, a genetic polymorphism affects our perception of androstenone. Molecular simulations totaling 0.12 ms predicted that, similarly to observations for other G‐protein‐coupled receptors with known experimental structures, an activation pathway connects the ligand and the G‐protein binding site. The 3D model activation mechanism correlates with in vitro data and notably predicts that the OR7D4 WM variant is not activated. Upon activation, an OR‐specific sequence motif is the convergence point of the mechanism. Our study suggests that robust homology modeling can serve as a powerful tool to capture OR dynamics related to smell perception.     

Modulating the Folding Landscape of Superoxide Dismutase 1 with Targeted Molecular Binders

Amyotrophic lateral sclerosis, or Lou Gehrig's disease, is characterized by motor neuron death, with average survival times of two to five years. One cause of this disease is the misfolding of superoxide dismutase 1 (SOD1), a phenomenon influenced by point mutations spanning the protein. Herein, we used an epitope‐specific high‐throughput screen to identify a peptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by a factor of 2.5. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics.