Assay Mixture Research Articles

Evaluation of the Effects of Some Metal Chlorides on the Initial Reaction Rate of Crude Peroxidase from Watermelon Peels

Aims: This study evaluates the effect of some chloride on the initial reaction rates of crude peroxidase from watermelon peels. Study Design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between April 2021 and June 2021. Methodology: The kinetics of crude peroxidase catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine (TMB) in the presence of varying concentrations of different chloride salts and hydrogen peroxidase was determined spectrophotometrically at 655nm. The assay mixture contained 2.3 mL of sodium phosphate buffer of pH 7.0, 0.1 mL of a crude enzyme from the watermelon peels, 0.2 mL of varying concentration of the respective chloride salts, 0.2 mL of 0.02 mM TMB, and 0.2 mL of 2 mM hydrogen peroxidase added last to start the reaction. Results: Results showed that chloride salts of Pb2+ Hg2+ Fe2+ and Na+ had peroxidase activating effects at low salt concentration (1 mM). The activating effect increased proportionately up to their respective optimum salt concentration. Further increment of salt concentration above their respective optimum value lead to a decrease in initial reaction rate of the crude enzyme. Within a chloride concentration range of 0.025 mM to 3 mM, the highest activating effect recorded was in the order Pb2+ > Fe2+ > Na+ > Hg2+.. Results show that Pb2+ had the highest enzyme activating effect at the lowest concentration (0.5 mM) of the salt when compared with other salts within the same concentration range. Conclusion: These findings are of great significance in research to understanding the mechanism of action of peroxidase from the peels of watermelon, especially as the search for cheap and alternative sources of peroxidases continues.

A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity

BackgroundDetection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy. ResultsWe developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5′-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h. Significance and noveltyDeveloped method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

Detection of c.375A>G, c.385A>T, c.571C>T, and sedel2 of FUT2 via Real-Time PCR in a Single Tube

α(1,2)fucosyltransferase (Se enzyme) encoded by FUT2 is involved in the secretor status of ABH(O) blood group antigens. The sedel2 allele is one of the non-functional FUT2 (se) alleles in which 9.3 kb, containing the entire coding region of FUT2, is deleted by Alu-mediated nonhomologous recombination. In addition to this allele, three SNPs of FUT2, c.375A>G, c.385A>T, and c.571C>T, appear to be prevalent in certain Oceanian populations such as Polynesians. Recently, we developed an endpoint genotyping assay to determine sedel2 zygosity, using a FAM-labeled probe for detection of the sedel2 allele and a VIC-labeled probe for the detection of FUT2. In this study, instead of the VIC probe, a HEX-labeled probe covering both c.375A>G and c.385A>T and a Cy5-labeled probe covering c.571C>T were added to the sedel2 allele assay mixture to allow for the simultaneous detection of these four variations via endpoint genotyping for sedel2 zygosity and fluorescence melting curve analysis for c.375A>G, c.385A>T, and c.571C>T genotyping. The results obtained from 24 Samoan subjects using this method were identical to those obtained using previous methods. Therefore, it appears that the present method can accurately determine these four variations simultaneously.

Label-free selective and sensitive colorimetric detection of ampicillin in milk and water using silver nanoparticles

Overuse of antibiotics like ampicillin in various developing and industrializing countries leads to the evolution of drug-resistant bacteria and different adverse effects on an individual's health. Though creating a facile, sensitive, and selective method for ampicillin detection is critical, this remains a strategic task. The poly(vinyl alcohol) (PVA)-capped silver nanoparticles (AgNPs) were used to develop a colorimetric assay for the detection of ampicillin. UV-Vis spectroscopy shows a strong peak at 402 nm due to the excitation of surface plasmon resonance (SPR). The X-ray diffraction (XRD) pattern of AgNPs shows lattice planes (111) and (200) correspond to 2θ values of 38.4° and 44.67°, respectively. Transmission electron microscopy (TEM) histogram shows the large number of particles synthesized in the range of 9–13 nm. As-synthesized AgNPs were used to evaluate the selectivity of the assay against various competitive antibiotics. The colour change of the assay mixture from yellow to orange was found in the presence of ampicillin due to target-specific aggregation and localized surface plasmon resonance changed from 402 nm to 550 nm. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM and 18.8 μM (linear range 0.5–10 μM) respectively. The colorimetric assay was used to detect the ampicillin-spiked actual samples (tap water, Yamuna River water, and milk). The developed colorimetric assay provides a real-time, rapid, and easy-to-use platform for detecting ampicillin for real sample analysis.

Ag@CDs nanohybrid: Fabrication, design of a multi-mode chemosensory probe for selective Fe3+ detection and logic gate operation

In this study, we propose a unique use of silver–carbon dot nanohybrid (Ag@CDs) with an average size of 16 nm as a multi-mode sensor for the selective detection of Fe3+ and the construction of logic gates based on these unique detection properties. The Ag NPs exhibit colourimetric sensing and fluorescence quenching in response to Fe3+ in the concentration range of 10–100 ppm, with the carbon dots acting as the fluorescent entity. Surprisingly, the nanohybrid was shown to have excellent sensitivity to Fe3+, resulting in aggregation and formation of yellowish-brown clumps. When the reaction mixture was treated with Fe3+, there was a discernible change in the colour of the assay mixture and fluorescence quenching. That is, the Ag@CDs acted as a calorimetric and fluorescence multi-mode sensor. Even in interfering groups in the natural river water sample, the produced nanohybrid displayed good selectivity towards Fe3+, with a minimum LOD of 0.76 ppm. Further, we constructed an advanced logic system, IMP-OR gate, by using additional inputs - ascorbic acid and urea.

Effects of Temperature on the Activity of Congregibacter litoralis KT 71 β -lactamase in the Hydrolysis of 4-nitrophenyl Dodecanoate

The effects of temperature on the activities of Congregibacter litoralis KT71 β-lactamase (CLL) was determined by varying the assay temperature from 30°C to 70°C. The enzyme’s residual activity was also determined by incubating the assay mixture which excluded the substrate for one hour at 70°C before addition of the substrate to start the reaction. The enzyme activity was measured using a spectrophotometer at a wavelength of 405 nm. The hydrolysis of 4-nitrophenyl dodecanoate to yield 4-nitrophenol was monitored by reading the absorbance at 25 minutes after the addition of substrate to the pre incubated assay mixture. The assay was done in three replicates. The initial velocity was determined and this was compared with Thermomyces lanuginosus Lipase (TLL) which served as a reference enzyme. Results show that increase in temperature from 30°C to 70°C proportionately decreased the activity of Congregibacter litoralis KT71 β-lactamase. Similarly, Thermomyces Lanuginosus Lipase showed a progressively decreasing activity as the temperature increased within the same range. Results from the assay on the residual activity at 70°C shows that both CLL and TLL has residual activities of 63% and 48% respectively. This suggests that reactions involving these enzymes at 70°C can occur without the enzyme completely inactivated. Results also shows that CLL is more thermally stable than TLL at 70°C. Considering the vast importance of thermally stable lipases and their industrial applications, there is need to study the kinetics of this enzyme to give an understanding of how the enzyme system works. Hence findings from this study is of great significance.

Effects of Chlorides of Lead and Some Transition Metals on the Kinetics of Crude Peroxidase from Watermelon Seeds

Aims: This study investigates the effect of chlorides of lead and some transition metals on the kinetics of crude peroxidase from watermelon seeds
 Study design: In vitro enzyme assay.
 Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between April 2021 and June 2021
 Methodology: The kinetics of crude peroxidase catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine (TMB) in the presence of varying concentrations of different chloride salts and hydrogen peroxidase was determined spectrophotometrically at 655nm. The assay mixture contained 2.3 mL of sodium phosphate buffer of pH 7.0, 0.1 mL of the crude enzyme from the seeds of watermelon, 0.2 mL of varying concentration of the respective chloride salts, 0.2 mL of 0.02 mM TMB, and 0.2 mL of 2 mM hydrogen peroxidase added last to start the reaction.
 Results: Results showed that except for nickel chloride, chloride salts of Pb2+, Hg2+, and Fe2+ had peroxidase activating effects. Mercury chloride and lead chloride proportionately increased the enzyme activity within a salt concentration range of 1.5 and 3 mM. In comparison, ferric chloride had an optimum concentration of 2.5 mM for peroxidase activation; mercury chloride had the highest peroxidase activation effect compared with chlorides of Pb, Ni, and Fe.
 Conclusion: These findings are of great importance to industries in understanding the mechanism of action of peroxidase from the seeds of watermelon, especially as the search for cheap and alternative sources of peroxidases continues.

Effects of Some Alkali and Alkaline Earth Metal Ions on the Initial Reaction Rates of Congregibacter litoralis KT71 β-lactamase Hydrolysis of 4-Nitrophenyl Myristate

The effects of some alkali metal ions (Na+ and K+) and alkaline earth metal ions (Mg 2+ and Ba2+) on the initial reaction rates of Congregibacter litoralis KT71 β-lactamase hydrolysis of 4-nitrophenyl myristate was investigated by varying the concentrations of the metal ions in the assay mixture which comprised of 100 µl of standard enzyme solution, 200 µl of varying concentration of metal ions, 500 µl of 50 mM sodium phosphate buffer pH 7.5 and 200 µl of 4-nitrophenyl myristate (substrate) which was added last to the assay mixture after an incubation time of 10 minutes at 44 oC. The enzyme activity was measured spectrophotometrically using a UV-780 recording spectrophotometer at a wavelength of 405 nm. The hydrolysis of 4-nitrophenyl myristate to yield 4-nitrophenol was monitored by reading the absorbance at 25 minutes. Results showed that the alkaline earth metal ions (Ba2+ and Mg2+) had higher enzyme activation effect than the alkali metal ions (K+ and Na+) Also, all metal ions except Mg2+ showed enzyme stimulatory effect at low concentrations (<2 mM) but inhibitory at higher ion concentrations (2 mM - 3 mM). Mg 2+ caused a proportionate decrease in enzyme activity from its peak (when metal ion concentration was lowest). Results from this research is of great significance to the industrialist especially where the search for novel lipases with unique characteristics suitable for the industries are inevitable.

EIF4B Is a Novel Target of CAMK2G and Promotes Proliferation of Malignant Cells in Myelofibrosis

BackgroundMyelofibrosis (MF) is a myeloproliferative neoplasm which is associated with megakaryocytic atypia, fibrosis in the bone marrow (BM), and extramedullary hematopoiesis, followed by progressive hematopoietic failure and leukemic transformation. JAK1/JAK2 inhibitors are currently available and reduce spleen volume, improve symptoms related to MF and prolong the overall survival (OS). Although the benefits associated with JAK1/JAK2 inhibitors are well established, not all patients respond to these inhibitors, and long-term exposure to these inhibitors results in the emergence of resistant clones based on the reactivation of the JAK-STAT pathway. Therefore, new therapeutic strategies targeting other molecules can provide additional benefits to patients with MF. In the previous study, we identified Calcium/Calmodulin Dependent Protein Kinase II Gamma (CAMK2G) as a new therapeutic target for MF by performing compound screening. Furthermore, CAMK2G inhibition can overcome drug-resistance against JAK2 inhibitors. Therefore, it is important to investigate the mechanisms underlying the therapeutic effect of CAMK2G inhibition. In this study, to explore the mechanism underlying the therapeutic effect of CAMK2G inhibition, we performed the immunoprecipitation mass spectrometry to find out the protein that has the direct interaction with CAMK2G.MethodsQuantitative Proteomic Analysis of the Target Proteins of CAMK2GThe protein complexes with FLAG-tagged CAMK2G (FLAG-CAMK2G) were trapped by anti-FLAG antibody. The eluted assay mixtures were reduced, alkylated and digested into peptides. Each peptide solution and control mixture were labeled with differential stable isotope tags. The samples were quantitatively analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The spectra were searched against the SWISS-PROT databases using SEQUEST on Proteome Discoverer software 2.2 (Thermo Scientific).ResultsTo reveal the , we conducted the immunoprecipitation assays by FLAG-CAMK2G or FLAG-tag alone, overexpressed in MF model cells. Because CAMK2G has kinase activity, we attempted to reveal the kinase-activity-dependent targets using unhydrolyzable ATP analog, AMP-PNP（5'-adenylyl-imidodiphosphate）that can maintain the strong-binding state of kinase with its target. The protein complexes with FLAG-CAMK2G were trapped by anti-FLAG antibody. After samples were digested into peptides and the candidate proteins were identified and quantitatively analyzed by mass spectrometry. As a result, we identified eukaryotic translation initiation factor 4B (eIF4B) as a protein that has a direct interaction with CAMK2G. eIF4B is a part of the complex involved in the initiation of translation. It has been reported that eIF4B plays a role in the translation of factors involved in anti-apoptosis (Bcl2, Bclxl, Mcl1), cell cycle (Cdc25c), and cell proliferation (c-Myc). We then checked whether knockdown of eIF4B decrease proliferation of MF model cell line. shRNA-mediated silencing of eIF4B decreased cell growth of these cells. Furthermore, the phosphorylation of eIF4B was increased by the ectopic expression of MPL W515L, one of the common mutations found in MF. Also, the phosphorylation of eIF4B was increased by the overexpression of CAMK2G. We then explored the proteins regulated by eIF4B in MF and identified that knockdown of eIF4B decreased the amount of Bcl2 protein.ConclusionIn our study, we performed immunoprecipitation mass spectrometry and identified eIF4B as a partner protein of CAMK2G. Since CAMK2G inhibition was shown to be effective against MF in vitro and in vivo, we focused on eIF4B as a potential effector in MF. We also showed that overexpression of MPL W515L and CAMK2G phosphorylates eIF4B and that knockdown of eIF4B inhibited proliferation of MF cells. Furthermore, Bcl2 can be one of the target proteins regulated by eIF4B. Based on these, eIF4B plays an important role in MF. We further perform ribosome profiling to comprehensively understand the regulation of translation by eIF4B. Our research not only elucidate the pathogenesis of MF but also identify new therapeutic targets for MF. DisclosuresMasamoto: MSD K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Kurokawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau.

A Study on Evaluation of Alkaline and Acid Phosphatase Isozymes During Different Developmental Stages of New Breeding Lines and Races of Bombyx mori L

It is interesting to note that different silkworm races reared in laboratory offer an important testing ground for the application of biochemical methods to taxonomic problems. Moreover, there is scarcity of knowledge on enzyme studies in new breeding lines and races of silkworm specially Bombyx mori L. Therefore, we designed the present study with the main goal to evaluate the activities of alkaline and acid phosphatases quantitatively during different developmental stages of new breeding lines and races viz. Kalimpong-A (KA), B18, Pure Mysore (PM), evolved R1 and R2 of Bombyx mori L. Quantitative estimations of in alkaline and acid phosphatases were expressed in terms of enzyme activity. Alkaline and acid phosphatase activities during the different developmental stages of KA, NB18, PM, evolved R1 & R2 races were determined using Sodium-1 naphthahyl phosphate as a substrate following the dye-coupling method. The assay mixture included 2 ml of substrate and 0.2 ml of enzyme extract and incubation was made for 30 minutes at 25°c. The reaction was stopped by adding 2 ml of post coupling solution (5 parts of 4% sodium dodecyl sulphate and 2 parts of 0.2% Fast red TR salt) and colorimetric determination were made at 540 nm. Results illustrated that the activity of phosphatases was found to be different and high activity was found in the larval stage, which is feeding stage followed by pupae.

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Interaction of Cucurbit[7]uril With Protease Substrates: Application to Nanosecond Time-Resolved Fluorescence Assays.

We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a “dark quencher.” The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster\u2019s Blue in methanol dehydrogenase assays

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV–Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster’s blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented.Graphic abstractLanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Optimization of Conditions for the Higher Level Production of Protease: Characterization of Protease from Geobacillus SBS-4S

Current study was planned keeping in mind the importance of proteases and their role in industry. In the present study protease was produced using Luria Bertani medium. The LB medium was supplemented with various carbon and nitrogen sources separately for the enhancement of growth of Geobacillus SBS-4S and for protease production. The optimization studies demonstrated the increase in protease production from 10.6 to 24.4 U/mL or 34.5 U/mL when the medium was supplemented with additional 2% yeast extract or 5% wheat bran respectively. Under the optimal conditions we could produce 46.2 U/mL of protease. The protein was purified by column chromatography and the purified protein was utilized for characterization studies. SDS-PAGE analysis confirmed the molecular weight of protease as 37 kDa. The enzyme exhibited its maximal activity at 60 °C and pH 9.0. Presence of Ca2+ and Mn2+ at a final concentration of I mM in the activity assay mixture enhanced protease activity from 100% to 133 and 150% respectively. Protease activity was slightly reduced (92%) in the presence of SDS whereas the presence of non-ionic detergents Triton X-100, Tween-20 and Tween-80 reduced the enzyme activity to 87, 82 and 69% respectively. Thermostability studies demonstrated that the protein was stable with 50% residual activity after an incubation of 2 h and 25 min at 60 °C in the presence of 1 mM Mn2+. The kinetic studies demonstrated the Km and Vmax values of 16.67 mg/mL and 143 U/mL respectively. The stability of protease at wide range of pH and temperature makes this enzyme suitable for its utilization in detergent and poultry feed industry.

Cloning and Overexpression of Strictosidine β-D-Glucosidase Gene Short Sequence from Catharanthus roseus in Escherichia coli.

Purpose: Strictosidine-β-D-glucosidase (SGD) is considered as a key enzyme in the production of bisindole alkaloids in Catharanthus roseus. The present study illustrated the production of a short sequence of this enzyme in Escherichia coli without codon optimization. Methods: Strictosidine-β-D-glucosidase (sgd) gene short sequence (1434 bp), which lacks the conserved sequence KGFFVWS and the localization peptide sequence at the C-terminal, was amplified from cDNA of C. roseus leaves, cloned and expressed in Escherichia coli. The activity of the produced protein in cell free lysate was tested using total alkaloid extract of C. roseus leaves. Results: HPLC and LC-MS analysis of the assay mixture revealed the disappearance of the strictosidine peak. Conclusion: SGD short sequence can be produced in Escherichia coli in active form without codon optimization.

Multimode sensing of riboflavin via Ag@carbon dot conjugates

In this study, we propose a simple and versatile tri-modal (colorimetric, fluorometric, and electrochemical) means for detection of riboflavin using silver carbon dot (Ag@CD) nanoconjugates. Hydroxyl-rich carbon dot (CD) synthesized by microwave-assisted method using citric acid and PEG was used as reductant for Ag@CD formation. Effect of CD and AgNO3 concentration on Ag@CD synthesis was examined. Furthermore, we observed that the Ag@CD conjugates were capable of selectively detecting riboflavin. The sensing mechanism was based on aggregation of nanoconjugates as confirmed from scanning electron microscope micrographs. Red shifting of surface plasmon resonance peaks to around 550 nm concomitant with quenching and red shift of fluorescence peak was observed. Colorimetric assay showed a change in colour of assay mixture to red and detection limit of 2.5 µM was observed. In addition, electrochemical detection showed a high selectivity for riboflavin even in the presence of interfering analytes with a sensitivity of 1.56 µA/mM/cm2. This rationally designed tri-modal sensing platform offers simplicity of operation and faster detection which may be developed into a point-of-care diagnostic device.

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Abstractω‐Transaminases (ω‐TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high‐throughput activity assay of ω‐TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω‐TA activities toward ketones in a 96‐well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω‐TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild‐type ω‐TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω‐TA activity was also demonstrated by supplementing the assay mixture with color‐developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω‐TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH‐based assay is expected to be promising for high‐throughput screening of enzyme collections and mutant libraries to fish out the best ω‐TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.

GPR31 and GPR151 are activated under acidic conditions.

Recent studies have revealed that not only proton-sensing channels, but also one family of G protein coupled-receptors (GPCRs) comprising OGR1, GPR4, G2A, and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31, and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31-Giα and GPR151-Giα fusion proteins in a narrow range between pH 6 and pH 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at approximately pH 5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

Development and comparative evaluation of two immunoassay platforms for bioanalysis of crizotinib: A potent drug used for the treatment of non-small cell lung cancer

This study describes, for the first time, the development of two platforms of competitive fluorescent immunoassays for bioanalysis of crizotinib (CZT), a potent drug used for the treatment of non-small cell lung cancer (NSCLC). These platforms were microwell-based heterogeneous fluoroimmunoassay (FIA) and a kinetic exclusion assay (KinExA) with KinExA™ 3200 immunosensor. Both FIA and KinExA were developed using same reagents; mouse anti-CZT antibody and a capturing reagent of CZT conjugated with bovine serum albumin (CZT-BSA). In the FIA, the CZT-BSA coated onto the microwells of the assay plate was present simultaneously in the assay mixture (CZT and its antibody). In the KinExA, the antibody was allowed to pre-equilibrate with CZT, and then the incubation mixture was rapidly passed through a microcolumn containing CZT-BSA coated onto polymethyl methacrylate (PMMA) beads. The analytical performances of both assays were comparatively evaluated in terms of assay working range, limit of detection, precision profile, and accuracy. The results revealed that KinExA yielded higher sensitivity and better precision than FIA; whereas, both assays had comparable accuracies. Both FIA and KinExA were superior to all the existing chromatographic methods for CZT in terms of the assay sensitivity, convenience, analysis throughputs. The proposed FIA and KinExA are anticipated to effectively contribute to the therapeutic drug monitoring (TDM) of CZT in clinical settings.

Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetics.

The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5'-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50 °C which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28%) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones.