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Fungal phytopathogens cause severe losses to crops worldwide and significantly reduce the quality and quantity of the produce. A shift towards reducing the usage of chemically synthesized pesticides by various biocontrol methods, strategies and approaches is observed in plant disease management. Symbiotic bacteria that live within the plant i.e. endophytes provide the host plant with many advantages in terms of metabolism and disease resistance. Bacterial antagonists play a significant role in controlling plant pathogens and diseases. In the present study 18 endophytic bacteria isolated from grape leaf were collected from ICAR-NRCG, Pune. In prospect to disease management, various endophytic bacteria were investigated for their antifungal activity against Sclerotium rolfsii, Macrophomina phaseolina, Alternaria spp., Fusarium spp. and Pythium spp. Dual culture antifungal activity indicated that 15 isolates have inhibitory effect against at least one of the tested pathogen. It was also observed that all the isolates have produced significant amount of hydrolytic enzymes i.e. cellulase, chitinase, amylase, protease and lipase, but the first two were the principal enzymes produced by them. These results provide a better understanding of endophytic bacteria in the control of pathogenic fungi and suggest their key role in the domain of biocontrol against diverse plant pathogens. [ABSTRACT FROM AUTHOR]

Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (Ips typographus). Despite these defences, the ESBB infests Norway spruce, causing significant ecological damage by exploiting weakened trees and using pheromones for aggregation. However, the mechanism of sensing and resistance towards host allelochemicals in ESBB is poorly understood. We hypothesised that the exposure of ESBB to spruce allelochemicals, especially monoterpenes, leads to an upsurge in the important detoxification genes like P450s, GSTs, UGTs, and transporters, and at the same time, genes responsible for development must be compromised. The current study demonstrates that exposure to monoterpenes like R-limonene and sabiene effectively elevated detoxification enzyme activities. The differential gene expression (DGE) analysis revealed 294 differentially expressed (DE) detoxification genes in response to R-limonene and 426 DE detoxification genes in response to sabiene treatments, with 209 common genes between the treatments. Amongst these, genes from the cytochrome P450 family 4 and 6 genes (CP4 and CP6), esterases, glutathione S-transferases family 1 (GSTT1), UDP-glucuronosyltransferase 2B genes (UDB), and glucose synthesis-related dehydrogenases were highly upregulated. We further validated 19 genes using RT-qPCR. Additionally, we observed similar high expression levels of detoxification genes across different monoterpene treatments, including myrcene and α-pinene, suggesting a conserved detoxification mechanism in ESBB, which demands further investigation. These findings highlight the potential for molecular target-based beetle management strategies targeting these key detoxification genes. [ABSTRACT FROM AUTHOR]

Fungi have been used to produce a number of bioactive compounds that include enzymes and antibacterial agents with unique characteristics in vitro. Penicillium chrysogenum is a mould with great potentials for producing important secondary metabolites. This study aimed at characterizing α-amylase and antimicrobial activity of P. chrysogenum. The organism was grown in a solid-state fermentation (SSF) medium using cassava peels as only carbon source. The molecular mass of the amylase was estimated to be 59 kDa by combined results of Amicon Ultra-15 filter unit (Sigma) 50 kDa MWCO and polyacrylamide gel electrophoresis (PAGE). Optimum activity of 93.1 U/mL was noticed at 30oC and pH of 5.8. Stability assay revealed that the enzyme was stable at 40°C and 50°C by retaining 66% and 37% residual activities, respectively. Also, crude extract of P. chrysogenum was found to inhibit Pseudomonas aeruginosa and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 25 and 12.5 mg/mL respectively. Therefore, these properties make P. chrysogenum an interesting fungus that has the potential of being used to produce both antibacterial agents and α-amylase for the bio-economy.

Diversifying wheat (Triticum aestivum L.)-based cropping systems is important for the sustainability of dryland agriculture. Research has focused on the agronomic benefits of increased crop diversity in semi-arid environments, but less is known about the impacts of increased crop diversity on the soil microbial community. This work compared soil health parameters between a continuous wheat crop sequence to a diverse sequence that included pea (Pisum sativum L.), proso millet (Panicum miliaceum L.), safflower (Carthamus tinctorius, L.), and spring wheat. Respiration was higher (p <.005) in the diverse sequence while activity of N-acetyl-β-d-glucosaminidase was lower (p <.05) with a mean rate of 26.3 and 16.3 mg ρ-nitrophenol kg−1 soil h−1 for the continuous wheat and diverse sequences, respectively. The mean net nitrogen mineralized during the growing season was 33.2 ± 2.5 kg ha−1 and was not different between treatments (p >.05). No difference was observed in bacterial alpha diversity, while fungal community diversity was 52% lower in the diverse rotation. The results of this work suggest that specific crops in a rotation may impact microbial processes related to nitrogen mineralization and that the soil fungal community may be more sensitive to changes in crop sequence than the soil bacterial community. [ABSTRACT FROM AUTHOR]

Introduction: Conventional practice in the management of acute TTP entails empirical treatment of suspected cases whilst awaiting confirmatory ADAMTS13 deficiency testing. Rapid ADAMTS13 assays offer increased accessibility and rapid diagnostics. The new automated HemosIL AcuStar® ADAMTS13 assay has seen increasing use among UK TTP Specialist Centres alongside the traditional ELISA method to confirm severe ADAMTS13 deficiency. Methods: A multi-centre retrospective case-control study was performed to review patients demonstrating discrepant ADAMTS13 activity results measured using rapid (AcuStar®) and ELISA assays in parallel from September 2019 to December 2021. Cases were compared with a cohort of suspected TTP patients exhibiting no difference in assay results and in relation to their presenting characteristics and pre-test probability of a diagnosis of TTP. Results: Where the clinical index of suspicion for TTP was high at presentation, acute TTP was confirmed using the AcuStar® assay < 0.2 IU/dL and subsequently < 10 IU/dL by ELISA with zero incidence of discrepancy. For patients with low clinical suspicion of acute TTP, a discrepancy between the AcuStar® and ELISA assay results was observed in 2% of cases; 5–10 IU/dL in AcuStar®, confirmed as >20 IU/dL by ELISA. A concurrent cancer diagnosis or sepsis was observed in 40% of discrepant cases. Conclusions: Where acute TTP is strongly suspected, there is a good correlation between the rapid AcuStar® ADAMTS13 assay and the conventional ELISA assay. Where the clinical suspicion of acute TTP is low, caution should be exercised in the interpretation of the ADAMTS13 activity using the AcuStar® assay. Accurate interpretation requires robust ADAMTS13 testing algorithms to be incorporated into diagnostic pathways. [ABSTRACT FROM AUTHOR]

Indoles and pyrroloindoles are structural motifs present in many biologically active natural products. Multiple classes of enzymes catalyze the transformation of indoles into pyrroloindoles via group transfer followed by intramolecular cyclization, such as peroxydases, methyltransferases, and prenyltransferases. Due to the selective introduction of a stereogenic center, these enzymes receive increasing attention as catalytic tools for the production of pharmacologically relevant compounds. Two new colorimetric assays are described in this work, which allow for the quantification of such enzymatic reactions from the perspective of the substrate and the product. For the substrates, the indole assay is based on a modified version of the Ehrlich test, with the use of light as a driving force for color formation. The pyrroloindole assay uses cerium sulfate as a reagent for the colorimetric quantification of the enzymatic products. The assays are complementary and both were successfully utilized for enzymatic activity determination of a C3‐indole methyltransferase. They can facilitate high‐throughput screening of mutant libraries, offering support for the engineering of such enzymes, but can also be used as stand‐alone methods for the detection and quantification of natural products. [ABSTRACT FROM AUTHOR]

Wheat germ acid phosphatase (WGAP) is a commonly used protein in laboratory settings for studying the release of inorganic phosphate (Pi) from phosphate esters under acidic conditions. When kinetically quantifying the enzyme, the typical procedure involves a spectrophotometric assay that indirectly measures WGAP activity. In this study, a new assay was developed to directly measure WGAP activity, involving the integration of a 31P NMR resonance peak representing the Pi product with a chemical shift of δ = 5.25 ppm. In order to compare the effectiveness of this new assay with the traditional method, both the 31P NMR spectroscopy-based assay and the traditional spectrophotometric-based assay were used side-by-side to evaluate WGAP activity. The extent to which activity was measured accurately using a UV-Visible spectrophotometer was quite limited, as the instrument only allowed accurate absorbance readings at relatively lower concentrations. Alternatively, performing analysis with 31P NMR spectroscopy introduced many advantages as the isotope is naturally abundant and the detection of the 31P signal at higher concentrations was possible. The total average WGAP specific activity determined from the spectrophotometric assays was 0.436 ± 0.023 μmol product/min/mg enzyme, and for the 31P NMR assays was 0.368 ± 0.098 μmol product/min/mg enzyme. When the two datasets of specific activity values from each method were compared, the analysis revealed a statistically significant similarity between the medians, thus confirming the viability of the new assay. These initial studies indicate that an assay based upon the integration of 31P NMR spectroscopy could be used for kinetic analysis of any reaction involving phosphate transfer. [ABSTRACT FROM AUTHOR]

Herbicide degradation is one of the key processes determining whether the use of herbicide will have any impact on environmental quality, and it is dependent on various biotic and abiotic factors. The present study evaluates the impact of various abiotic factors such as soil type, application rate, incubation temperature, soil pH, soil microorganisms and sunlight on persistence of metribuzin and its effect on soil enzymes. Half-lives (DT50) varied significantly with application rate of metribuzin and physicochemical properties of soil and ranged from 15.17 to 46.59 days. Metribuzin degraded 2.2 to 6.5 times slower in sterilised soil (DT50 = 35.21 to 115.60 days) as compared to unsterilised soil, indicating microbial degradation is one the most predominant contributors towards degradation of metribuzin in soil. Increase in degradation rate on exposure to sunlight indicated photodegradation also contributes significantly to the degradation of metribuzin. Deaminometribuzin, diketometribuzin and deaminodiketometribuzin metabolites of metribuzin were detected in all treatments. Dehydrogenase and alkaline phosphatase activities were significantly affected by application of metribuzin while urease activity remained unaffected. The physicochemical properties viz. pH, organic matter content and temperature should be considered along with application rate of metribuzin in order to achieve satisfactory weed control and reduce environmental risk associated with the use of metribuzin in different crops. [ABSTRACT FROM AUTHOR]

Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of l-arginine to l-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against l-arginine as its substrate in a reaction at 20 °C, pH 9. At 10–35 °C and pH 7–9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Despite modern-day technology, food waste is still a major concern. Bread is one of the major consumable foods in Asian countries and has also been observed as waste in various institutions. Fermentation is one of the most socioeconomic methods of utilizing various types of raw materials including biomass as waste. The present work highlights the production of enzymes pectinase and amylase using a Bacillus strain (BIOSMNF02) and beard waste as raw material under minimal water content. Among all the physio-chemical parameters attempted in the minimum water content, the optimum result was obtained in a medium having a moisture level of 1:2.25 (w/v) at pH 7, with the inoculum percentage as 4% (w/v). The optimum fermentation period for the maximum pectinase and amylase production was found to be on the 5th day of culturing condition and the 10th day of fermentation respectively. The maximum activity of enzymes was found to be at 37℃, pH 7, and substrate concentration of 200 µg/mL of pectin and 300 µg/mL of starch for pectinase and amylase respectively. The selected bacterium, Bacillus strain (BIOSMNF02) was isolated for its pectinase production capability, but it also showed the synthesis of amylase that offered novelty in the present study. [ABSTRACT FROM AUTHOR]

Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (Ips typographus). Despite these defences, the ESBB infests Norway spruce, causing significant ecological damage by exploiting weakened trees and using pheromones for aggregation. However, the mechanism of sensing and resistance towards host allelochemicals in ESBB is poorly understood. We hypothesised that the exposure of ESBB to spruce allelochemicals, especially monoterpenes, leads to an upsurge in the important detoxification genes like P450s, GSTs, UGTs, and transporters, and at the same time, genes responsible for development must be compromised. The current study demonstrates that exposure to monoterpenes like R-limonene and sabiene effectively elevated detoxification enzyme activities. The differential gene expression (DGE) analysis revealed 294 differentially expressed (DE) detoxification genes in response to R-limonene and 426 DE detoxification genes in response to sabiene treatments, with 209 common genes between the treatments. Amongst these, genes from the cytochrome P450 family 4 and 6 genes (CP4 and CP6), esterases, glutathione S-transferases family 1 (GSTT1), UDP-glucuronosyltransferase 2B genes (UDB), and glucose synthesis-related dehydrogenases were highly upregulated. We further validated 19 genes using RT-qPCR. Additionally, we observed similar high expression levels of detoxification genes across different monoterpene treatments, including myrcene and α-pinene, suggesting a conserved detoxification mechanism in ESBB, which demands further investigation. These findings highlight the potential for molecular target-based beetle management strategies targeting these key detoxification genes.

Here, we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3-acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base-exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base-exchange activity. [ABSTRACT FROM AUTHOR]

Fungi have been used to produce a number of bioactive compounds that include enzymes and antibacterial agents with unique characteristics in vitro. Penicillium chrysogenum is a mould with great potential for producing important secondary metabolites. This study aimed to characterize α-amylase and antimicrobial activity of P. chrysogenum. The organism was grown in a solid-state fermentation (SSF) medium using cassava peels as the only carbon source. The molecular mass of the amylase was estimated to be 59 kDa by combined results of Amicon Ultra-15 filter unit (Sigma) 50 kDa MWCO and polyacrylamide gel electrophoresis (PAGE). The optimum activity of 93.1 U/mL was noticed at 30°C and pH of 5.8. A stability assay revealed that the enzyme was stable at 40°C and 50°C by retaining 66% and 37% residual activities, respectively. Also, crude extract of P. chrysogenum was found to inhibit Pseudomonas aeruginosa and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 25 and 12.5 mg/mL respectively. Therefore, these properties make P. chrysogenum an interesting fungus that has the potential of being used to produce both antibacterial agents and α-amylase for the bio-economy. [ABSTRACT FROM AUTHOR]

The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RTqPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management. [ABSTRACT FROM AUTHOR]

Catalase is a key antioxidative enzyme, and a deficiency or malfunction of catalase is hypothesized to be related to various diseases. To investigate catalase activity, it is important to use reliable methods and experimental protocols enabling consistent fallouts. One major problem, however, is that the activity values obtained with different techniques and procedures can vary to a large extent. The aim of this work was to identify experimental conditions that provide similar catalase activity values with two different methods based on either spectrophotometry or chronoamperometry. The investigated parameters include the concentration of catalase and its substrate (H2O2), as well as the effect of deoxygenation of the catalase medium by nitrogen (N2). Within the frame of investigated conditions, we show that spectrophotometry is strongly affected by the catalase concentration, whereas chronoamperometry is shown to be more dependent on the substrate concentration. Deoxygenation leads to elevated catalase activity values in the case of chronoamperometry, whereas it shows no influence on the results obtained with spectrophotometry. In particular, in the case of low substrate concentrations (i.e., low catalase reaction rates), higher and more accurate results are obtained with deoxygenation in the case of chronoamperometry measurements due to minimized oxygen escape. The effect of deoxygenation, giving rise to elevated catalase activity values, however, is not statistically significant at high substrate concentrations, implying that the protocol can be simplified by excluding this step as long as the other parameters are optimized. Finally, by comparing the two methods at different experimental conditions, we identified protocols resulting in similar results, i.e., 10 mM H2O2 and catalase activity of 4–5 U/mL. Based on this work, improved consistency of catalase activity data obtained with different methodologies and in different labs is expected.

Background and Aim: Caspase-9 is a key enzyme in the intrinsic pathway of apoptosis that its activity is regulated by various mechanisms such as phosphorylation. It has been reported that phosphorylation of serine 310 in murine caspase-9 prevents enzyme processing. The role of this residue in human caspase-9 activity in not clear. In this study we investigated the effect of negative charge on serine 310 in caspase-9 activity. Materials and Methods: Considering that phosphorylation leads to negative charge on caspase-9, the codon of serine 310 in human capsase-9 was mutated to aspartate via quick change site-directed mutagenesis. Recombinant wild type and mutant caspase-9 were expressed in BL21(DE3) and purified by affinity chromatography. The temperature profile and activity of the mutant caspase-9 were assessed by chromogenic substrate of Ac-LEHD-pNA in vitro, and compared to those of wild enzyme. Student’s t test was used for data analysis. Results: The results showed that kinetics parameters of S310D mutant and wild type caspase-9 were similar, but their temperature profiles were different. Comparison of S310D mutant enzyme and wild type caspase-9 showed that S310D mutant enzyme had higher activity at 37 oC and lower activity at 4, 15, 45 and 60 oC. Conclusion: In our study, the negative charge on serine 310 in caspase-9 led to change in the profile temperature of the enzyme with no effect on kinetic parameters.

In the last decade, research has shown that most diseases are associated with organelle dysfunction in which metabolites play a crucial role or indicate specific processes. Peroxisomes are cellular organelles attracting an increasing amount of attention and are now recognized as essential players in physiological conditions and diseases. However, a limited amount of research focuses on isolating the organelles and studying their properties and the diseases resulting from organelle dysfunction. All methods for isolating peroxisomes are based on fresh tissue samples. To the best of our knowledge, this is the first work in which peroxisomes have been isolated from frozen rat liver. In our work, we isolated peroxisomes from frozen rat liver at −80 °C and evaluated the separation success and degree of purification of isolated peroxisomes by measuring the relative specific activity, purification fold, and percentage yield (Y%) of organelle marker enzymes in the isolated fractions. The percentage of protein distribution and density was also estimated. Our results showed that the purified peroxisome fraction (F3-peroxisome) had significantly higher relative specific activity, as well as the highest purification fold and percentage yield of catalase compared with the enzyme markers of other organelles in the postnuclear supernatant (PNS), postmitochondrial supernatant (PMS), and light mitochondria–peroxisome (LM-P) fractions. In addition, the percentage of protein distribution was significantly lower in the F3-peroxisome fraction compared with PNS, PMS, and LM-P fractions while the percentage of protein distribution and density of the F3-peroxisome fraction after iodixanol centrifugation were significantly higher than those of the F1 and F2 fractions. The present work demonstrates the possibility of isolating peroxisomes from frozen liver samples efficiently, which could pave the way for further research in the future on other subcellular organelles from frozen samples. [ABSTRACT FROM AUTHOR]

With advancements in bionanotechnology, the field of nanobiocatalysts has undergone rapid growth and revolutionized various nanomaterials as novel and fascinating nanocarriers for enzyme immobilization. Nanotubes, nanofibers, nanopores, nanoparticles, and nanocomposites have been successfully developed and used as nanocarriers. The construction of robust nanobiocatalysts by combining enzymes and nanocarriers using various enzyme immobilization techniques is gaining incredible attention because of their extraordinary catalytic performance, high stability, and ease of reusability under different physical and chemical conditions. Creating appropriate surface chemistry for nanomaterials promotes their downstream applications. This review discusses enzyme immobilization on nanocarriers and highlights the techniques, properties, preparations, and applications of nanoimmobilized enzymes. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H2O2 and dehydroascorbate concentrations. The applicability of the assay was demonstrated. [ABSTRACT FROM AUTHOR]

Background: Wildfire severity mediates key dynamics, such as nutrient pulses, that regulate the recovery of ecosystem functioning. Large shifts in vegetation communities associated with plant invasions are often coupled with changes in soil communities; thus, it's critical to understand how fire severity may interact with vegetation type and soil communities to mediate ecosystem recovery. Methods: Following a 2017 wildfire in southern California, soils from areas dominated by native coastal sage scrub or exotic annual grasses that experienced a low or high severity fire event were analyzed for nutrient concentrations and two proxies for ecosystem function—microbial respiration and enzymatic activity potentials over the first-year post-fire. Aims: We predicted that increasing fire severity would positively correlate with soil nutrient concentrations. Thus, higher severity burned soil would experience a greater downregulation of enzyme activity as potential microbial nutrient limitation was alleviated, a relationship that would be stronger in shrub dominated soil. Results: We observed a strong soil nitrogen (N) pulse post-fire, which was greatest in shrub dominated soil; however, dominant vegetation had a variable effect on microbial responses. Enzyme activities were downregulated in CSS soil, but the grass dominated soil response was inconsistent. After 1 year, soil N remained elevated in burned soil, suggesting that basal soil N concentrations were altered. Conclusions: Persistent, residual soil N concentrations are of particular concern in high fire risk ecosystems, which will likely experience increasing fire frequency associated with environmental change; thus, encouraging the regrowth of opportunistic vegetation in subsequent growing seasons will be key to minimize long-term changes to these ecosystems. [ABSTRACT FROM AUTHOR]

Here, we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3-acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base-exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base-exchange activity.

ω-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.

Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H2O2 and dehydroascorbate concentrations. The applicability of the assay was demonstrated.

Controlling post-prandial hyperglycemia and hyperlipidemia, particularly by regulating the activity of digestive enzymes, allows managing type 2 diabetes and obesity. The aim of this study was to assess the effects of TOTUM-63, a formulation of five plant extracts (Olea europaea L., Cynara scolymus L., Chrysanthellum indicum subsp. afroamericanum B.L.Turner, Vaccinium myrtillus L., and Piper nigrum L.), on enzymes involved in carbohydrate and lipid absorption. First, in vitro inhibition assays were performed by targeting three enzymes: α-glucosidase, α-amylase, and lipase. Then, kinetic studies and binding affinity determinations by fluorescence spectrum changes and microscale thermophoresis were performed. The in vitro assays showed that TOTUM-63 inhibited all three digestive enzymes, particularly α-glucosidase (IC50 of 13.1 µg/mL). Mechanistic studies on α-glucosidase inhibition by TOTUM-63 and molecular interaction experiments indicated a mixed (full) inhibition mechanism, and higher affinity for α-glucosidase than acarbose, the reference α-glucosidase inhibitor. Lastly, in vivo data using leptin receptor-deficient (db/db) mice, a model of obesity and type 2 diabetes, indicated that TOTUM-63 might prevent the increase in fasting glycemia and glycated hemoglobin (HbA1c) levels over time, compared with the untreated group. These results show that TOTUM-63 is a promising new approach for type 2 diabetes management via α-glucosidase inhibition. [ABSTRACT FROM AUTHOR]

L–DOPA decarboxylase (DDC) deficiency causes serious progressive neurodegenerative disorders. Developing a sensitive enzyme activity monitoring and inhibitors or activators screening approach is highly on demand for DDC deficiency diagnosis. Here, we report a simple and sensitive fluorescence supramolecular tandem assay based on reporter pair of cucurbit[7]uril and acridine orange for DDC activity monitoring continuously. Furthermore, this strategy demonstrates the potential of STA to screen inhibitors of DDC. [ABSTRACT FROM AUTHOR]

Lanthanide complexes have a range of photophysical properties – large pseudo‐Stokes shifts, narrow linewidths, long emission lifetimes – that make them ideal as emissive probes. These same properties also render them excellent donors in assays relying on Förster resonance energy transfer (FRET). Over the last 20 years these assays have increased in sophistication so that a range of biological applications has now been demonstrated. These range from easy‐to‐use quantitation of proteins (a practical alternative to western blotting) to sophisticated experiments which give real‐time information on the movement of proteins or allow simultaneous readout of both agonist and co‐activator binding to a protein. We introduce the principles and difficulties of using such assays before surveying the full scope of what they can achieve and attempting to anticipate future developments in this area. [ABSTRACT FROM AUTHOR]

Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography, ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293T cells, this method revealed a reduction in activity of 90, 36, and 96%, respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure–function relationships for ASNS variants reported for ASNSD patients. [ABSTRACT FROM AUTHOR]

Aim/Background: Pancreatic lipase (PL) inhibition has been suggested to be an effective method for obesity management. In this study, 10 novel thiazole-benzimidazole conjugates were designed. Materials and Methods: Conjugates were initially screened via in silico experiments, such as ADMET analysis and molecular docking, to identify the most promising PL inhibitors. Results: Results revealed that compounds 3, 6 and 8 had the most optimum druglikeness properties, and the highest binding affinity to PL, with binding energies of -7.7, -7.5 and -8.1 kcal/mol, respectively. Therefore, these promising derivatives were then synthesized and subjected to an in PL inhibition assay to validate the results of the in silico experiments. The synthetic compounds were fully characterized via FTIR, 1H-NMR, 13C-NMR and LCMS. Results of the enzymatic assay revealed that 3, 6 and 8 inhibited PL potently with high inhibition rates of greater than 80% at the highest tested dose, and demonstrated IC50 values of 68.53, 54.97 and 50.09 µM, respectively. Compound 8 was the most active derivative and displayed comparable activity to orlistat which possessed an IC50 value of 39.18µM. Conclusion: Therefore, we report the discovery of compound 8 as a highly potent PL inhibitor that could act as a lead compound to develop novel anti-obesity agents. [ABSTRACT FROM AUTHOR]

During the present research, 11 gut bacteria were isolated from the freshwater fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/ml pectinase and through molecular characterization the SS6 strain was identified as Aeromonas guangheii. During the culture of SS6 strain, a set of parameters were optimized through the response surface methodology with a Box–Behnken design, for the production of the enzyme. The optimal conditions were found to be 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 °C at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/ml. The purified pectinase's molecular weight was determined to be ~ 50 kDa (by 10% 2-D PAGE). Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and its nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, which was found to be over expressed in Escherichia coli BL21. The ORF encoded for a mature protein comprising of 425 amino acids (1236 nucleotides) with a predicted molecular weight of ~ 48.7 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes. [ABSTRACT FROM AUTHOR]

Abstract Background The present study focuses on the isolation of Bacillus thuringiensis bacterium from the gut of fresh water fish, Systomus sarana, the innovative optimization of culture parameters to produce maximum protease enzyme, by the isolated bacterium, and the elucidation of peptide profile of the protease. And the experimental data and results were authenticated through the response surface method (RSM) and Box-Behnken design (BBD) model. Results During the RSM optimization, the interaction of the highest concentrations (%) of 2.2 maltose, 2.2 beef extract, and 7.0 pH, at 37 °C incubation, yielded a maximum protease enzyme of 245 U/ml by the fish gut-isolated, B. thuringiensis. The spectral analysis of the obtained enzyme revealed the presence of major functional groups at the range of 610–3852 cm−1 viz., alkynes (–C≡C–H: C–H stretch), misc (P-H phosphine sharp), α, β-unsaturated aldehydes, and through PAGE analysis, its molecular weight was determined as 27 kDa. The enzyme’s MALDI-TOF/MS analysis revealed the presence of 15 peptides from which the R.YHTVCDPR.L peptide has been found to be a major one. Conclusions The fish gut-isolated bacterium, B. thuringiensis, SS4 exhibited the potential for high protease production under the innovatively optimized culture conditions, and the obtained result provides scope for applications in food and pharmaceutical industries.

Lysosomal acid lipase deficiency (LAL-D) (OMIM: 278000) is a lysosomal storage disorder with two distinct disease phenotypes such as Wolman disease and cholesteryl ester storage disorder (CESD), characterized by an accumulation of endocytosed cholesterol in the body. Due to the presence of multiple lipases in DBS, previous studies measured LAL enzyme activity in the presence of Lalistat-2, an established LAL-specific inhibitor (Hamilton J et al Chim Clin Acta (2012) 413:1207–1210). Alternatively, a novel substrate specific for LAL has been reported very recently (Masi S. et al Clin Chem (2018) 64:690–696). In this study, we examined the LAL enzyme activity of a Japanese population with the LAL-specific substrate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based enzyme assay whether an affected individual can be identified among this population. To achieve this, we first performed assay validation using LC-MS/MS. Under our experimental setting, typically we obtained LAL enzyme activity for QC High (100% enzyme activity) as 261.9 ± 3.2 μmol/h/L (n = 5) and for QC Low as (5% enzyme activity) as 14.7 ± 0.5 μmol/h/L (n = 5). The percentage of coefficient of variation for interday assay for QC High was 9.6% (n = 4) and for QC Low was 7.9% (n = 4), respectively. Based on these results, we further examined the LAL enzyme activity of control Japanese population and that of affected individuals with Wolman disease and CESD. The averaged enzyme activity for control newborns, Wolman, and CESD was 123.9 ± 53.9 μmol/h/L (n = 131), 6.6 ± 0.9 μmol/h/L (n = 3), and 4.8 ± 0.3 μmol/h/L (n = 3), respectively. These results suggest that an LAL-D-affected individual can be readily identified by enzyme activity using LC-MS/MS-based technique.

New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP3 to IP8, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP3-4K from C. neoformans (CnArg1) at low µM IC50s, but not IP3-4K from C. albicans (CaIpk2) and many analogues exhibited selectivity for CnArg1 over the human equivalent, HsIPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP3-4K. [ABSTRACT FROM AUTHOR]

Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective strategies in obesity treatment is the inhibition of PNLIP, which is possible to be achieved by specific phenolic compounds occurring in high abundance in some plants. In this study, a multidisciplinary approach is presented investigating the PNLIP inhibitory effect of 33 plants belonging in the Asteraceae botanical family. In the first stage of the study, a rapid and cost-efficient PNLIP assay in a 96-microwell plate format was developed and important parameters were optimized, e.g., the enzyme substrate. Upon PNLIP assay optimization, aqueous and dichloromethane Asteraceae plant extracts were tested and a cut-off inhibition level was set to further analyze only the samples with a significant inhibitory effect (inhibitory rate > 40%), using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) method. Specifically, a metabolomic suspect screening was performed and 69 phenolic compounds were tentatively identified, including phenolic acids, flavonoids, flavonoid-3-O-glycosides, and flavonoid-7-O-glycosides, amongst others. In the case of aqueous extracts, phytochemicals known for inducing PNLIP inhibitory effect, e.g., compounds containing galloyl molecules or caffeoylquinic acids, were monitored in Chrysanthemum morifolium, Grindella camporum and Hieracium pilosella extracts. All in all, the presented approach combines in vitro bioactivity measurements to high-end metabolomics to identify phenolic compounds with potential medicinal and/or dietary applications. [ABSTRACT FROM AUTHOR]

• A 2,668-bp cDNA encoding a full-length SuSy4 form (designated KC-SuSy), corresponding to SUS I group gene family, was isolated and characterized from a potato cultivar Kufri Chipsona-1. • The crucial domains/motifs and a three-dimensional (3-D) structure of KC-SuSy were predicted. • A total of 6 SUS genes were identified from the available potato genome sequence databases, and their probable biochemical characteristics were predicted. • Expression patterns of the SUS genes were examined by bioinformatics and experimental approaches. • SuSy activity was determined at various stages of tuberization. Sucrose synthase (SuSy, EC 2.4.1.13) refers to a glycosyltranferase (GT) that plays a crucial role in sugar metabolism mainly in the sink tissues of plants. Using uridine diphosphate (UDP) or adenosine diphosphate (ADP), SuSy catalyzes the reversible cleavage of sucrose into fructose, and UDP-glucose (UDP-Glc) or ADP-glucose (ADP-Glc), respectively. In plants, SuSy is encoded by relatively small gene families. It exists in multiple forms distributed differentially in the plant tissues suggesting their functional specializations. SuSy activity is regulated by a hierarchy of mechanisms including transcriptional control. Here, we report isolation and characterization of a 2,668-bp cDNA encoding a distinct full-length SuSy4 form, corresponding to SUS I group gene family, from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR using tuber total RNA. The predicted protein consisted of 805 amino acids (designated KC-SuSy). All of the available potato SuSy proteins could be categorized into 3 groups i.e., SUS I−III as reported earlier in other plants. Phylogenetic analysis was carried out using the SuSy sequences from the potato, tomato and Arabidopsis. Multiple sequence alignments revealed segment-wise identity and divergence between the SuSy forms. Using KC-SuSy sequence, secondary structures, crucial domains/motifs and amino acids, and a three-dimensional (3-D) structure were predicted. KC-SuSy was found to be predominantly expressed in tuber. The early stages of tuber development were associated with high levels of SuSy activity. This report would be particularly useful for further studies on the SuSy proteins in potato and other Solanaceae family members. [ABSTRACT FROM AUTHOR]

The search for new anti-inflammatory drugs with less side effects requires simple, fast and reliable screening methods. In this context, we have developed a sensitive thin-layer chromatographic (TLC) assay on silica gel plates to detect cyclooxygenase-2 (COX-2) inhibition. COX-2 catalyzes two sequential enzymatic reactions: a first oxygenation step that converts arachidonic acid into prostaglandin G2, and a subsequent reduction of prostaglandin G2 into prostaglandin H2. Our test is based on the co-oxidation during this peroxidation step of a co-substrate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), leading to a blue-grey product. As a consequence, COX-2 inhibitors appear on the TLC plate after revelation as clear spots against the colored background. Parameters such as concentrations of enzyme, substrate, and chromogenic reagent have been optimized. The limit of detection was found to be below the microgram for standard COX-2 inhibitors such as celecoxib or ibuprofen. The developed TLC assay was also conclusive when applied to 60 various natural pure compounds and some complex natural extracts. Results demonstrated a COX-2 inhibitory activity mostly for triterpene and sterol derivatives. This COX-2 TLC assay appears as a suitable low-cost and reliable strategy for the screening of natural extracts to discover new anti-inflammatory compounds. [ABSTRACT FROM AUTHOR]

Background: Lysosomal storage disorders (LSDs) are a heterogeneous family of genetic diseases with a broad phenotypic spectrum. There is a paucity of data on LSDs from developing countries. Objective: We aimed to study the pattern, relative frequency, and phenotypic spectrum of LSDs in children at an Egyptian medical center. Patients and Methods: This study included children < 18 years with LSDs diagnosed and followed up at an Egyptian medical center from January 2018 to December 2021. Data were collected on patients’ demographics, clinical features, characteristic metabolites, specific enzyme assay, and genetic testing. Results: Forty-two children (62% males, 74% parental consanguinity and 26% positive family history) were diagnosed with 10 different LSDs, representing 14% of all cases with inborn errors of metabolism (IEMs). The most frequent LSDs groups were mucopolysaccharidosis (MPS) (52.4%) and sphingolipidosis (40.8%). The most common individual diseases were MPS I (26.2%), Gaucher disease (23.8), MPS III (16.7%), and acid sphingomyelinase deficiency (11.9%). The median age at presentation was two years with a median diagnostic delay of 12 months. The most common clinical manifestations were delayed development, intellectual disability, visceromegaly, coarse facial features, and skeletal abnormalities. Finally, genetic data were available for only 12 patients (8 Gaucher disease, 3 MPS-III, and 1 MPS-VI). Conclusion: LSDs (most commonly MPS and Gaucher disease) represent an important part of IEMs at our medical center, and the diagnosis seems challenging and often delayed. [ABSTRACT FROM AUTHOR]

Bacterial pneumonia is considered one of the most virulent diseases with high morbidity and mortality rates, especially in hospitalized patients. Moreover, bacterial resistance increased over the last decades which limited the therapy options to carbapenem antibiotics. Hence, the metallo-β-lactamase-producing bacteria were deliberated as the most deadly and ferocious infectious agents. Sulphadiazine-ZnO hybrids biological activity was explored in vitro and in vivo against metallo-β-lactamases (MBLs) producing Klebsiella pneumoniae. Docking studies against NDM-1 and IMP-1 MBLs revealed the superior activity of the 3a compound in inhibiting both MBLs enzymes in a valid reliable docking approach. The MBLs inhibition enzyme assay revealed the remarkable sulphadiazine-ZnO hybrids inhibitory effect against NDM-1 and IMP-1 MBLs. The tested compounds inhibited the enzymes both competitively and noncompetitively. Compound 3b-ZnO showed the highest antibacterial activity against the tested metallo-β-lactamase producers with an inhibition zone (IZ) diameter reaching 43 mm and a minimum inhibitory concentration (MIC) reaching 2 µg/mL. Sulphadiazine-ZnO hybrids were tested for their in vitro cytotoxicity in a normal lung cell line (BEAS-2Bs cell line). Higher cell viability was observed with 3b-ZnO. Biodistribution of the sulphadiazine-ZnO hybrids in the lungs of uninfected rats revealed that both [124I]3a-ZnO and [124I]3b-ZnO hybrids remained detectable within the rats' lungs after 24 h of endotracheal aerosolization. Moreover, the residence duration in the lungs of [124I]3b-ZnO (t1/2 4.91 h) was 85.3%. The histopathological investigations confirmed that compound 3b-ZnO has significant activity in controlling bacterial pneumonia infection in rats. [ABSTRACT FROM AUTHOR]

Introduction: Histone modifying enzymes include several classes of enzymes that are responsible for various post-translational modifications of histones such as methylation and acetylation. They are important epigenetic factors, which may involve several diseases and so their assay, as well as screening of their inhibitors, are of great importance. Herein, a bioassay based on terbium-to-quantum dot (Tb-to-QD) time-resolved Förster resonance energy transfer (TR-FRET) was developed for monitoring the activity of G9a, the euchromatic histone-lysine N-methyltransferase 2. Overexpression of G9a has been reported in some cancers such as ovarian carcinoma, lung cancer, multiple myeloma and brain cancer. Thus, inhibition of this enzyme is important for therapeutic purposes. Methods: In this assay, a biotinylated peptide was used as a G9a substrate in conjugation with streptavidin-coated ZnS/CdSe QD as FRET acceptor, and an anti-mark antibody labeled with Tb as a donor. Time-resolved fluorescence was used for measuring FRET ratios. Results: We examined three QDs, with emission wavelengths of 605, 655 and 705 nm, as FRET acceptors and investigated FRET efficiency between the Tb complex and each of them. Since the maximum FRET efficiency was obtained for Tb to QD705 (more than 50%), this pair was exploited for designing the enzyme assay. We showed that the method has excellent sensitivity and selectivity for the determination of G9a at concentrations as low as 20 pM. Furthermore, the designed assay was applied for screening of an enzyme inhibitor, S-(5’-Adenosyl)-L-homocysteine (SAH). Conclusion: It was shown that Tb-to-QD FRET is an outstanding platform for developing a homogenous assay for the G9a enzyme and its inhibitors. The obtained results confirmed that this assay was quite sensitive and could be used in the field of inhibitor screening.

Abstract Monoamine oxidase A (MAO‐A) deficiency is a rare inborn error of metabolism with impaired degradation of biogenic amines including 5‐hydroxytryptamine (5‐HT), resulting in borderline intellectual disability and behavioral abnormalities. Genetic variants in MAOA need functional confirmation to enable a definite diagnosis. To this end, we developed an inexpensive, simple and nonradioactive MAO‐A activity assay based on the conversion of 5‐HT into 5‐hydroxyindoleacetic acid (5‐HIAA). Fibroblast cell lysates were incubated with 5‐HT and aldehyde dehydrogenase to allow 5‐HIAA production. 5‐HIAA was quantified using high‐performance liquid chromatography with fluorimetric detection. We optimized reaction mixture components, pH, and substrate concentration and tested linearity and specificity of the assay. We verified the functional validity of the enzyme assay using fibroblasts of controls, female mutation carriers and MAO‐A deficient patients. This included a newly described patient with a novel MAOA variant (c.1336G>A, p.(Glu446Lys)), who represents the fifth MAO‐A deficiency family so far. The optimized enzyme assay showed good linearity and specificity. Application to clinical samples showed a 100% differentiation of affected patients (with negligible MAO‐A enzyme activity) and controls or mutation carriers. In conclusion, the described MAO‐A activity assay is easy to implement and can readily be used to test the pathogenicity of variants in the MAOA gene in a clinical setting. Especially in this era of whole‐exome (and whole‐genome) sequencing, this functional assay fulfills a clinical need for functional confirmation of a suspected diagnosis of MAO‐A deficiency.

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC50 = 0.32 μmol/L; WS-635: IC50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.

In the last decade, research has shown that most diseases are associated with organelle dysfunction in which metabolites play a crucial role or indicate specific processes. Peroxisomes are cellular organelles attracting an increasing amount of attention and are now recognized as essential players in physiological conditions and diseases. However, a limited amount of research focuses on isolating the organelles and studying their properties and the diseases resulting from organelle dysfunction. All methods for isolating peroxisomes are based on fresh tissue samples. To the best of our knowledge, this is the first work in which peroxisomes have been isolated from frozen rat liver. In our work, we isolated peroxisomes from frozen rat liver at −80 °C and evaluated the separation success and degree of purification of isolated peroxisomes by measuring the relative specific activity, purification fold, and percentage yield (Y%) of organelle marker enzymes in the isolated fractions. The percentage of protein distribution and density was also estimated. Our results showed that the purified peroxisome fraction (F3-peroxisome) had significantly higher relative specific activity, as well as the highest purification fold and percentage yield of catalase compared with the enzyme markers of other organelles in the postnuclear supernatant (PNS), postmitochondrial supernatant (PMS), and light mitochondria–peroxisome (LM-P) fractions. In addition, the percentage of protein distribution was significantly lower in the F3-peroxisome fraction compared with PNS, PMS, and LM-P fractions while the percentage of protein distribution and density of the F3-peroxisome fraction after iodixanol centrifugation were significantly higher than those of the F1 and F2 fractions. The present work demonstrates the possibility of isolating peroxisomes from frozen liver samples efficiently, which could pave the way for further research in the future on other subcellular organelles from frozen samples.

Ribonuclease A (RNase A) plays significant roles in several physiological and pathological conditions and can be used as a valuable diagnostic biomarker for human diseases such as myocardial infarction and cancer. Hence, it is of great importance to develop a rapid and cost-effective method for the highly sensitive detection of RNase A. The significance of RNase A assay is further enhanced by the growing attention from the biotechnology and pharmaceutical industries to develop RNA-based vaccines and drugs in large part as a result of the successful development of mRNA vaccines in the COVID-19 pandemic. Herein, we report a label-free method for the detection of RNase A by monitoring its proteolytic cleavage of an RNA substrate in a nanopore. The method is ultra-sensitive with the limit of detection reaching as low as 30 fg per milliliter. Furthermore, sensor selectivity and the effects of temperature, incubation time, metal ion, salt concentration on sensor sensitivity were also investigated. [Display omitted] • A label-free nanopore single-molecule sensing system for detection of RNase A activity was developed. • The sensor was selective and highly sensitive with LOD reaching 30 fg per milliliter. • Temperature, incubation time, metal ion, salt concentration and especially salt gradient affected sensor sensitivity. [ABSTRACT FROM AUTHOR]

This study focuses on dilution effect of target-site resistance (TSR) to acetolactate synthase (ALS) inhibitors in Schoenoplectiella juncoides , which harbors two ALS genes, ALS1 and ALS2. We assessed gene expression, enzyme activity, and whole-plant resistance profiles across four S. j uncoides lines: the susceptible line, the parental resistant lines with a homozygous mutation in either ALS1 or ALS2 , and the bred progeny line with homozygous mutations in both ALS1 and ALS2. Gene expression and enzyme function showed a proportional relationship that the expression ratios of ALS1 to ALS2 , approximately 70:30, were consistent with the functional ratio predicted by the double-sigmoidal plateau positions observed in enzyme assays. However, at the whole-plant level, resistance did not correlate to the putative abundance of susceptible enzyme, but the parental lines showed similar resistance to each other despite different enzyme-level resistances. This suggests a non-proportional mechanism in the reflection of physiological enzymatic profiles to whole-plant resistance profiles. These findings highlight the complexity of herbicide resistance and the need for further research to understand the mechanisms that influence resistance outcomes. Understanding these relationships is essential for developing strategies to manage herbicide resistance effectively. [Display omitted] • ALS1 to ALS2 expression ratio was around 70:30. • Enzyme activity was proportional to the expression ratio. • Enzyme activity was diluted by a contamination of susceptible enzymes. • Verified dilution effect at whole-plant level with various ALS inhibitors. • Gene expression non-proportionally reflect to whole-plant resistance. [ABSTRACT FROM AUTHOR]

As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. We herein describe a U DG- RE tarded C RISPR A mplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10−4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance. [Display omitted] • URECA (U DG- RE tarded C RISPR A mplification assay) was developed for UDG assay. • Combination of isothermal amplification and CRISPR maximized signal amplification. • URECA is one-pot, isothermal, rapid (50 min), and sensitive (9.17 x 10−4 U/mL). • URECA can detect UDG in biological matrices and screen potential inhibitors. [ABSTRACT FROM AUTHOR]

We herein present a novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction. The detection probe employed as a key component in this technique serves as a substrate for RNase H and triggers the PS-THSP reaction upon the RNase H-mediated degradation of the probe. As a consequence, a large number of long concatemeric amplification products could be produced and used to identify the RNase H activity through the fluorescence signals produced by the nucleic acid-specific fluorescent dye, SYTO 9. Importantly, the use of the gp32 protein allowed the PS-THSP reaction to be performed at 37 °C, ultimately enabling an isothermal one-step RNase H assay. Based on this sophisticated design principle, the RNase H activity was very sensitively detected, down to 0.000237 U mL−1 with high specificity. We further verified its practical applicability through its successful application to the screening of RNase H inhibitors. With its operational convenience and excellent analytical performance, this technique could serve as a new platform for RNase H assay in a wide range of biological applications. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates. [ABSTRACT FROM AUTHOR]

To secure a sustainable food supply for the rapidly growing global population, great efforts towards a plant-based diet are underway. However, the use of plant proteins comes with several challenges, such as improvement or removal of undesired flavours, and generation of desired texture properties. Fermentation holds large potential to alter these properties, but compared to dairy fermentations, our knowledge on strain properties in different plant-based substrates is still limited. Here, we explored different lactic acid bacteria for their ability to grow, produce flavour compounds, or remove off-flavour compounds from different plant proteins. For this, 151 LAB strains from dairy and non-dairy origins were cultured in plant protein plus coconut oil emulsions supplemented with glucose. Pea, chickpea, mung, fava, and soybean proteins were used in the study and bacterial strains for screening included the genera Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc. Efficient, high throughput, screening on plant proteins was developed and strains were assessed for their ability to (i) acidify and decrease the pH; (ii) express key enzymes involved in the formation of amino acid derived flavours, which included PepN (aminopeptidase N), PepXP (X-prolyl dipeptidyl peptidase), EstA (esterase), BcAT (branched chain aminotransferase), CBL (cystathione beta lyase), and ArAT (aromatic aminotransferase); and (iii) improve the overall aroma profile by generating dairy/cheesy notes and decreasing off flavours. Suitable screening conditions were determined, and highlighted the importance that a sufficient heat treatment must be applied to samples containing plant proteins, prior to fermentation, as an outgrowth of spore forming Bacillus cereus was observed if the material was only pasteurised. Enzyme activities for strains measured in rich broth vs. a buffered protein solution showed little-to-no correlation, which illustrated the importance of screening conditions to obtain predictive enzyme measurements. Aroma formation analysis allowed to identify strains that were able to increase key aromas such as diacetyl, acetoin, 2- and 3-methyl butanol, and 2,3-pentanedione, as well as decrease the off-flavours hexanal, pentanal, and nonanal. Our findings illustrate the importance of strain specific differences in the assessed functionalities and how a methodical approach to screening LAB can be applied to select suitable microorganisms that show promise in fermentation of plant proteins when applied in non-dairy cheese applications. [ABSTRACT FROM AUTHOR]