Research on Some Freshwater Fish Catalase Activity - As a Potential Biomarker for Environmental Pollution

In the last decade studies show that water pollution is a very serious problem, especially since the degree of water pollution plays a key role in the growth and fish multiplication. As a biomarker of environmental pollution, antioxidant enzymes such as catalase (CAT; EC 1.11.1.6) play an essential role in preventing the harmful effects of heavy metals in the tissues of fish. These researches were carried out to investigate the effect of various environment conditions and some pollutant agents on oxidative stress in some aquatic organisms. The enzymatic assay of CAT (in fish organs, e.g., liver, kidney, gill, intestine and brain, as well as in mixture of these organs) was carried out according to standard enzyme assay protocol. The results showed decrease of CAT activity: the enzymatic activity was 35.89 ± 1.02 µmol H2O2/min/mg protein to pH 7 and 6.59 ± 0.47 µmol H2O2/min/mg protein to pH 12. More, the enzymatic activity was 38.1 ± 0.3 µmol H2O2/min/mg protein to pH 3. Also, the catalase activity increased with 23 enzymatic units to a less dissolved oxygen concentration (6.2877 mg/L). Furthermore, this study indicated that heavy metals, such as Cr, Cu and Zn can inhibit biochemical reactions in various organs of fish. During exposure duration of the fish to a mixture of metal ions the catalase activity decreases from 35.89 ± 1.02 µmol H2O2/min/mg protein to 23.51 ± 2.85 µmol H2O2/min/mg protein. On the other hand, as a result of the response of the enzyme system, the catalase activity increases to 36.25 ± 3.22 µmol H2O2/min/mg protein.

PRESTO-Tango Assay v2

PRESTO-Tango Assay protocol for assessing GPR68 activation by benzodiazepines at acidic pH.

Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription–free exponential amplification reaction, RTF-EXPAR

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription–free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

The Ames assay is the standard assay for identifying DNA-reactive genotoxic substances. Multiple formats are available and the correct choice of an assay protocol is essential for achieving optimal performance, including fit for purpose detection limits and required screening capacity. In the present study, a comparison of those parameters between two commonly used formats, the standard pre-incubation Ames test and the liquid-based Ames MPF™, was performed. For that purpose, twenty-one substances with various modes of action were chosen and tested for their lowest effect concentrations (LEC) with both tests. In addition, two sources of rat liver homogenate S9 fraction, Aroclor 1254-induced and phenobarbital/β-naphthoflavone induced, were compared in the Ames MPF™. Overall, the standard pre-incubation Ames and the Ames MPF™ assay showed high concordance (>90%) for mutagenic vs. non-mutagenic compound classification. The LEC values of the Ames MPF™ format were lower for 17 of the 21 of the selected test substances. The S9 source had no impact on the test results. This leads to the conclusion that the liquid-based Ames MPF™ assay format provides screening advantages when low concentrations are relevant, such as in the testing of complex mixtures.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

ATP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine

Microbial contamination may represent a loss of money for wine producers as several defects can arise due to a microorganism’s growth during storage. The aim of this study was to implement a bioluminescence assay protocol to rapidly and simultaneously detect bacteria and yeasts in wines. Different wines samples were deliberately contaminated with bacteria and yeasts at different concentrations and filtered through two serial filters with decreasing mesh to separate bacteria and yeasts. These were resuscitated over 24 h on selective liquid media and analyzed by bioluminescence assay. ATP measurements discriminated the presence of yeasts and bacteria in artificially contaminated wine samples down to 50 CFU/L of yeasts and 1000 CFU/L of bacteria. The developed protocol allowed to detect, rapidly (24 h) and simultaneously, bacteria and yeasts in different types of wines. This would be of great interest for industries, for which an early detection and discrimination of microbial contaminants would help in the decision-making process.

ULI-NChIP assay protocol

This protocol presents ULI-NChIP-seq (ultra-low-input micrococcal nuclease-based native ChIP-seq) assay to generate high quality and complexity genome-wide histone mark profiles from rare oocytes andembryos populations. The procedure of ULI-NChIP-seq assay typically consists of five parts including Binding antibodies to magnatic beads, Chromatin shearing and nuclear membrane solubilization, Magnetic immunoprecipitation, Washes and DNA isolation. Sample preparation involves to remove the zona Pellucida of oocyte and polar body to avoid the genomic contamination of polar bodies.

VaNGuard assay protocol for SARS-CoV-2 detection

This protocol presents the Variant Nucleotide Guard (VaNGuard) assay, which is robust towards viral mutations and can be performed on purified RNA or directly on nasopharyngeal (NP) swab samples. The procedure typically comprises three parts, namely sample preparation, RT-LAMP reaction, and Cas12a-based detection via fluorescence or lateral flow assay. Sample preparation from NP swabs involves Proteinase K digestion followed by heat inactivation. Purified RNA or digested NP swab samples are then added as templates into RT-LAMP reactions and incubated at 65ºC for 22 minutes. Next, enAsCas12a and ssDNA-probes are added and the reactions are incubated at 60ºC for another 5 minutes. End-point fluorescence can be detected by a plate reader or a real-time PCR machine. Alternatively, a lateral flow strip can be inserted into each reaction tube for equipment-free read-out. The VaNGuard assay is a rapid and convenient point-of-care test for SARS-CoV-2 and is applicable to resource poor settings.

Design Optimization of Centrifugal Microfluidic "Lab-on-a-Disc" Systems towards Fluidic Larger-Scale Integration

Enhancing the degree of functional multiplexing while assuring operational reliability and manufacturability at competitive costs are crucial components to enable comprehensive sample-to-answer automation, e.g., for use in common, decentralized “Point-of-Care” or “Point-of-Use” scenarios. This paper demonstrates a model-based ‘digital twin’ approach which efficiently supports the algorithmic design optimization of exemplary centrifugo-pneumatic (CP) dissolvable-film (DF) siphon valves towards larger-scale integration (LSI) of well-established “Lab-on-a-Disc” (LoaD) systems. Obviously, the spatial footprint of the valves and their upstream laboratory unit operations (LUOs) have to fit, at a given radial position prescribed by its occurrence in the assay protocol, into the locally available disc space. At the same time, the retention rate of rotationally actuated valve and, most challenging, its band width related to unavoidable experimental tolerances need to slot into a defined interval of the practically allowed frequency envelope. A set of design rules, metrics, and methods and instructive showcases for computationally assisted optimization of valve structures are presented.