Commentary regarding: “Activity determination of FAD‐dependent glucose dehydrogenase immobilized in PEDOT: PSS‐PVA composite films for biosensor applications”

In the light of continuous improvement and optimization, recent experiments that the authors conducted give new insights into the applied evaluation method of Riegel et al. [1]: Thorough investigations of the previous results regarding the usage of the Lowry Assay showed discrepancies in the determination of the released amount of protein in the analysis solution. The accurate quantification of this parameter is crucial as it directly influences the calculation of the residual enzymatic activity. In concrete terms, this finding has a major impact on the presented and discussed results in the article “Activity determination of FAD‐dependent glucose dehydrogenase immobilized in PEDOT: PSS‐PVA composite films for biosensor applications” [1]. Thus, this commentary addresses the new insights concerning the applied evaluation method, explains necessary revisions and discusses new conclusions derived from the adjusted evaluation method.     

Spectroscopic characterization,DFT studies,molecular docking and cytotoxic evaluation of 4‐nitro‐indole‐3‐carboxaldehyde: A potent lung cancer agent

The 4‐nitro‐1H‐indole‐carboxaldehyde (NICA) molecule was characterized experimentally using FT‐IR, FT‐Raman and UV‐Vis spectra, and it was studied theoretically using DFT calculations. The optimized structure of the NICA molecule was determined by DFT calculations using B3LYP functional with cc‐pVTZ basis set. The electron localization function (ELF) and local orbital localizer (LOL) studies were performed to visualize the electron delocalization in the molecule. The experimental and theoretical wavenumbers of the title molecule were assigned using VEDA 4.0 program. The charge delocalization and stability of the title molecule were investigated using natural bond orbital (NBO) analysis. Frontier molecular orbitals (FMOs) and related molecular properties were calculated. UV‐Vis spectrum was calculated theoretically and validated experimentally. The reactive sites of the molecule were studied from the MEP surface and Fukui function analysis. The molecular docking analysis reveals that the NICA ligand shows better inhibitory activity against RAS, which causes lung cancer. The in vitro cytotoxic activity of the molecule against human lung cancer cell lines (A549) was determined by MTT assay. Thus, the NICA molecule can be used as a potential candidate for the development of the drug against lung cancer.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Cell‐mediated immunity and multi‐locus heterozygosity in bluethroat nestlings

Recent evidence suggests that marker‐based heterozygosity‐fitness correlations may be driven by only one or a few markers, indicating local heterozygosity effects caused by linkage disequilibrium with functional genes. In this study, we investigated the relationship between microsatellite heterozygosity and a measure of cell‐mediated immunity (phytohaemagglutinin; PHA) in bluethroat (Luscinia s. svecica) nestlings using a full‐sibling design. We found significant positive associations between PHA response and two different indices of microsatellite heterozygosity, i.e. multi‐locus heterozygosity and mean d². However, model comparisons disclosed that both associations were more likely caused by local effects rather than general effects and that the two local effects appeared to be realized through two different genetic mechanisms. Our results indicate that both the random assortment of parental chromosomes during meiosis as well as inbreeding can drive heterozygosity‐fitness correlations.     

Replication of IMR‐32‐adapted JC virus clones in human embryonic kidney cells

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT‐PCR assay revealed that large T antigen expression in cells transfected with IMR‐32‐adapted JCVs was significantly greater than in those transfected with Mad‐1 or CY. DNA replication assay and viral load verified that the IMR‐32‐adapted JCVs were replication‐competent in 293 cells, but not Mad‐1 or CY JCVs. These results suggest that a 293 culture system with IMR‐32‐adapted JCVs may be a useful tool for assessing replication of JCV in vitro.     

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies by up‐converting phosphor technology‐based lateral‐flow assay

In this study, an up‐converting phosphor technology‐based lateral‐flow (UPT‐LF) assay was developed to detect severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies rapidly and specifically. SFTSV recombinant N protein (SFTSV‐rNP) was coated on analytical membrane for sample capture, up‐converting phosphor (UCP) particles were used as the reporter, the luminescence emitted by UCP particles was converted to a measurable signal by a biosensor. The performance of UPT‐LF assay was evaluated by testing 302 field serum samples by ELISA (enzyme‐linked immunosorbent assay), Western blotting and UPT‐LF assay. UPT‐LF assay exhibited a lower detection limit than ELISA, and a satisfied level of agreement was exhibited by Kappa statistics (Kappa coefficient = 0.938). Considering Western blotting as the reference for comparison, the sensitivity and specificity of UPT‐LF assay could reach 98.31% and 100%. UPT‐LF assay showed no specific reaction with hantavirus total serum antibodies, which avoids the misdiagnosis of SFTSV from hantavirus that could cause similar clinical symptoms. UPT‐LF assay was able to achieve acceptable results within 15 min and needed only 10 μL sample for each test. As a whole, UPT‐LF assay is a candidate method for on‐site surveillance of SFTSV total antibodies owing to its excellent sensitivity, specificity, stability, easy operation and for being less time consuming.     

Development of a chemiluminescent imaging assay for the detection of anti‐erythropoietin antibody in human sera

Measuring low amounts of anti‐erythropoietin antibodies (anti‐EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high‐throughput chemiluminescent (CL) imaging assay was developed for the detection of anti‐EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12–13.91 ng/mL. The limit of detection (LOD, 3σ) for the CL‐imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme‐linked immunosorbent assay (ELISA), the LOD of the CL‐imaging assay is 50‐fold lower. The recoveries of anti‐EPO Abs in the fortified serum were in the range 87.1–116.9% using the present method, which highlighted the validity of the CL‐imaging assay system to accurately determine the anti‐EPO Abs in serum samples. CL‐imaging assay was used to evaluate the presence of anti‐EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti‐EPO Abs. Copyright © 2008 John Wiley & Sons, Ltd.     

Functional Roles of N‐Linked Glycosylation of Human Matrix Metalloproteinase 9

Matrix metalloproteinase‐9 (MMP‐9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP‐9 has two well‐defined N‐glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N‐glycosylation‐deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism‐dependent miRNA‐mediated inhibitory mechanism. hMMP‐9 cDNA encoding amino acid substitutions at residues 38 (modified‐S38, mS38) or 120 (N120S) were created in the background of a miRNA‐binding site disrupted template and expressed by transient transfection. hMMP‐9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP‐9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non‐secreted variants and co‐immunoprecipitation confirmed an enhanced strong interaction between the non‐secreted hMMP‐9 and the ER‐resident protein calreticulin (CALR). Removal of N‐glycosylation at residue 38 revealed an amino acid‐dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N‐glycosylation strongly regulates hMMP‐9 secretion. This is mediated, however, through a novel mechanism of cloaking an N‐glycosylation‐independent strong interaction with the ER‐resident CALR.      

Development of taxon‐specific markers for high‐throughput screening of microbial‐feeding nematodes

In an effort to assess the taxonomic identity of large‐scale samplings of nematodes from the Konza Tallgrass Prairie, we sequenced a portion of the 18S rRNA gene and its associated internally transcribed spacer (ITS1) from 74 nematodes encompassing four taxonomic families. From these sequences, we have developed a series of molecular probes to distinguish 16 distinct microbivorous nematode taxa. Using a combination of low power microscopy and taxon‐specific real‐time probes, the 74 nematodes were correctly assigned to their respective taxonomic groups. This optimized method provides a high‐throughput assay to determine nematode identities across larger data sets.     

Streamlined Development of Targeted Mass Spectrometry‐Based Method Combining Scout‐MRM and a Web‐Based Tool Indexed with Scout Peptides

MS‐based targeted proteomics is a relevant technology for sensitive and robust relative or absolute quantification of proteins biomarker candidates in complex human biofluids or tissue extracts. Performing a multiplex assay imposes time scheduling of peptide monitoring only around their expected retention time that needs to be defined with synthetic peptide. Time‐scheduled monitoring is clearly a constraint that precludes from straightforward assay transfer between biological matrices or distinct experimental setup. Any unexpected retention time (RT) shift challenges assay robustness and its implementation for large‐scale analysis. Recently, Scout‐multiple reaction monitoring that fully releases multiplexed targeted acquisition from RT scheduling by successively monitoring complex transition groups triggered with sentinel molecules called Scout has been introduced. It is herein documented how Peptide Selector database and tool streamlines the building of a multiplexed method thanks to RT indexation relative to Scout peptides. This case study deals with surrogate peptides of biomarker candidates related to drug‐induced liver and vascular injury, running such on‐line built method (eight Scouts triggering the monitoring of a total of 692 transitions) enables 100% recovery of a panel of 93 spiked‐in heavy labeled standards, despite significant RT shifts between serum, plasma, or urine. This result illustrates the simplicity of automatically building and deploying robust proteomics targeted assay.     

Identification of acetylcholinesterase inhibitors using homogenous cell‐based assays in quantitative high‐throughput screening platforms

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell‐based assays in high‐throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10‐acetyl‐3,7‐dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH‐SY5Y). An enzyme‐based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high‐throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.     

A novel colorimetric assay of β‐D‐glucans in basidiomycete strains by alcian blue dye in a 96‐well microtiter plate

Basidiomycete strains synthesize several types of β‐d ‐glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these β‐d ‐glucans in mushroom strains is of great biochemical importance. Because published assay methods for these β‐d ‐glucans present some disadvantages, a novel colorimetric assay method for β‐d ‐glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (～14 nm) in UV‐Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high‐throughput colorimetric assay method on microtiter plates was used for quantification of β‐d ‐glucans in the range of 0–0.8 μg, with a slope of 44.15 × 10^?2 and a limit of detection of 0.017 μg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for β‐1,3‐d ‐glucan. The present assay method exhibited a 10‐fold higher sensitivity and a 59‐fold lower limit of detection compared with the published method with congo red. β‐d ‐glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify β‐d ‐glucans from other biological sources. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1526–1535, 2015     

Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation

Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro‐ and anti‐apoptotic Bcl‐2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high‐content high‐throughput screen to identify small molecules which inhibit the cellular process of Bax re‐distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post‐induction of cisplatin‐mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mitochondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin‐induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax‐mediated PCD.