The over use of pharmaceutical compounds, essentially antibiotics, led to an increase of their concentrations in aquatic ecosystems. In this study, the white-rot fungus Coriolopsis gallica (high-laccase-producing fungus) was investigated for the biodegradation of ampicillin (AMP) under different cultivation conditions. The biotransformation of the antibiotic was confirmed by means of high-performance liquid chromatography (HPLC), and the antibacterial activity was evaluated via the bacterial growth inhibition agar well diffusion method using Escherichia coli as an ampicillin-sensitive test strain. The results showed that AMP (50 mg L-1) was totally removed by C. gallica after 6 days of incubation in a liquid medium. The antibiotic removal rate was maximal with a fungal culture aged 9 days. Such a culture achieved the removal of a higher concentration of up to 500 mg L-1 of ampicillin in 3 days. This higher antibiotic removal rate was concomitant with the maximal laccase production in culture supernatant. In addition, four consecutive doses of 500 mg L-1 of ampicillin were transformed by the same fungal culture in 24 days. After that, the fungus was unable to remove the antibiotic. The measurement of ligninolytic enzyme activity showed that laccase of C. gallica might participate in the biotransformation of AMP.

Thromboembolic conditions, such as stroke and myocardial infarction [MI], are the primary causes of mortality globally. High efficiency was demonstrated by fibrinolytic enzymes isolated from microbial sources in dissolving various blood clots. The study aimed to investigate and produce bacterial fibrinolytic enzymes from various Egyptian soil conditions using recombinant DNA technology. Fifty soil samples were examined to determine the production of fibrinolytic enzymes. The plasmin plate technique assessed the thrombolytic activities of the strong fibrinolytic enzyme-producing bacterial isolates. In addition to determining the molecular mass using a mass spectrometer and the western blot method, the activity and thermostability of the enzymes were characterized. An affinity chromatography approach was used to purify the fibrinolytic enzymes. However, bacterial recombinant DNA technology produced the pure bacterial fibrinolytic enzyme. Several dosage formulations for the formulation of fibrinolytic enzymes were tested. By using affinity chromatography and the SDS-PAGE test, the isolated bacterial fibrinolytic enzyme in this investigation was almost 83% pure. Bacillus cereus ATCC 4342 was identified as the predominant bacterial isolate secreting fibrinolytic enzymes. It was discovered that the fibrinolytic enzyme activity of fifty soil samples ranged from 0.167 U/ml to 1.853 U/ml. The extracellular enzyme extracted from Bacillus cereus culture supernatant demonstrated a relatively stable fibrinolytic activity over 18 hours at a pH range of 7.4–10, as well as stability with maximum productivity at 43℃ for over 60 minutes. In this investigation, the enzyme was referred to as Fibrin-proteinase[ FP]. The fibrin clots were directly thrombolyzed by the FP. The calculated values for the specific activity and molecular mass of the FP were approximately 40.71 units/mg of protein and approximately 30 KDa, respectively. It was observed that the presence of Fe+3 marginally boosted protease activity, but the presence of EDTA decreased it. On the other hand, adding monovalent cations like K+1 and Na+1 significantly increased the fibrinolytic activity of FP. The enzyme exhibited activity throughout the temperature range of 20 to 65℃, peaking at 55℃. The ideal range for the generation of protease was 35–43℃ and 8.0–10. The thermostable fibrinolytic enzyme FP, which was isolated from Bacillus cereus ATCC 4342 in various soil environments in Egypt during the current study, was a promising fibrinolytic enzyme because it demonstrated a more extensive and effective fibrinolytic activity and fewer side effects than the fibrinolytic enzymes that are currently available worldwide. It is advised that further research be done on optimizing the right formulation of bacterial fibrinolytic enzymes in the future to help dissolve different types of blood clots and, as a result, lower the high death rate that exists worldwide.

Soil extracellular enzyme activity (EEAs) and enzymatic stoichiometry (ES) can provide a crucial indication of changes in soil ecosystem's nutrient availability and the microbial resource limitations. However, the changing characteristics of soil EEAs and ES at different stages of the native succession process and their key drivers are unclear. In order to investigate the soil EEAs, ES and driving factors of soil under vegetation at different succession stages, we adopted the "spatio-temporal substitution" method to collect the surface soil of bryophyte community, herbaceous community, shrub community and tree community in the new volcanic lava platform of Wudalianchi Volcanic Nature Reserve. We measured seven soil EEA, including carbon(C)-acquiring enzyme (β-1,4-glucosidase (BG)), N-acquiring enzymes (β-N-acetyl-glucosaminidase (NAG) and leucine aminopeptidase (LAP)) and phosphorus (P)-acquiring enzyme (acid phosphatase (AP)) activities. The length and angle of vectors defined by ratios of enzyme activities (BG/(NAG + LAP) vs. BG/AP) were used to indicate relative microbial investments in C- (length), and N- and P- (angle) acquiring enzymes. Our results showed that the contents of TC, TN, TP, MBC, DOC and NO3-N in shrub community soil were significantly higher than those in bryophyte, herb and tree communities, and increased by 441%, 246%, 137%, 5570%, 12% and 484%, respectively. The highest soil EEA of C-, N- and P-acquiring were found in shrub community, and the soil EEAs/MBC of C-, N- and P-acquiring were the highest in bryophyte community. Enzyme C:N, C:P and N:P ratios increased progressively in the order of bryophyte, herb and shrub community, but the enzyme C:N, C:P and N:P ratios of tree community were both far less than shrub community. Vector lengths increased progressively in the order of bryophyte (1.16), herb (1.27), tree (1.29) and shrub (1.40), and Vector angles decreased progressively in the order of bryophyte (49.15°), herb (45.65°), Tree (45.31°) and shurb (44.54°), suggested that as succession progresses, soil microbial nutrients transforms from P limitation (angle＞45°) to N limitation (angle＜45°). Redundancy analysis showed that TC, TN, EC and C:N were important drivers of variation in soil EEAs and ES in vegetation at different succession stages. Our findings highlight that the primary succession process cause nutrient limitation transformation. Soil ES might be a sensitive indicator mediated by soil microorganisms to the relative resource limitation at different stages of the primary succession process.

The carbohydrate antigen Sda is expressed on cells and secretions of the vast majority of Caucasians. The epitope is formed by a terminal GalNAc residue β4-linked to a α3-sialylated galactose. Different carbohydrate chains N- or O-linked to glycoproteins can be terminated by this epitope. The final step of Sda biosynthesis is catalyzed by the GalNAc transferase B4GALNT2. In this review, we will discuss the multifaceted aspects of B4GALNT2/Sda biology. In particular, we will see its implications in fertility and pregnancy, in susceptibility to infectious diseases, in chronic kidney diseases and in Duchene muscular dystrophy. In particular, we will show its regulation and prognostic implications in cancer.

Simple Summary: The distribution of typical bacterial redox enzymes such as pyruvate-ferredoxin oxidoreductase (PFOR) in protozoa remains interestingly puzzling. Previous studies have demonstrated diverse cellular localizations of PFOR in some amitochondriate anaerobic protozoa. PFOR is of particular pharmacological importance because it catalyzes the reductive bio-activation of nitro-based prodrugs to cytotoxic radical metabolites. Metronidazole was developed primarily as an antiprotozoal agent against infections caused by Trichomonas vaginalis. However, its antimicrobial spectrum was subsequently expanded to cover anaerobic bacterial infections. It has been shown that mutations in the genes encoding PFOR result in inherent resistance of PFOR-possessing anaerobic protozoa and bacteria to nitro-based prodrugs. Deciphering the evolutionary history of PFOR is crucial in deepening our understanding of the evolution of anaerobic pathogens and unfold new approaches for drug discovery and targeting in pathogen chemotherapy. Abstract: The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative bioinformatics approaches to test the hypothesis that PFOR was most likely acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other genes that were putatively acquired using proteome-wide and gene expression analyses. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance optimal adaptation of protozoa in hostile environments. The exclusively expressed proteins obtained from Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, showed an over-expression of eleven genes involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide a more validated evidence to support the horizontal transfer of pfor from bacteria.

Glycerol represents a useful functional component of heat-transfer fluids. Glycerol is known to be triboelectrically active. We report how the flow of glycerol, stopped in a cylindrical coiled pipe, influences horseradish peroxidase (HRP) enzyme. Namely, glycerol flow was pumped through the coil and then stopped. After that, we incubated the 0.1 µM solution of HRP near the side of the coil, and then adsorbed HRP onto mica. This operation markedly increased the HRP aggregation on mica in comparison with the control sample. We explain this phenomenon by the influence of triboelectrically induced electromagnetic fields. Our results can be useful in the development of flow-based systems for biosensors and bioreactors.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif locates next to another, ill-defined cysteine that we show in-here is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCM) suggests a candidate Fe-S cluster ligand role. Hence, we generated by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Conventional fertilization practices can lead to many ecological problems, such as nutrient imbalance, soil acidity, and reduced soil fertility in natural rubber plantations. To address these challenges, a field investigation was strategically carried out to substitute inorganic fertilizer with organic fertilizers consisted of six treatments, i.e., no fertilization (CK), inorganic fertilizer (NPK), 25 % replacement of inorganic through organic (25%M), 50 % replacement of inorganic through organic (50%M), 75 % replacement of inorganic through organic (75%M), and 100% organic fertilizer (100%M). The soil physicochemical properties (SOC, TN, AN, C:N, pH, NH4+-N, and NO3--N) and carbon and nitrogen converting enzymes (BG, NAG, and LAP) were all determined. The partial substitution of inorganic fertilizers with organic fertilizers (i.e., 75%M at surface soil layer) showed higher SOC (14.52 g.kg-1), TN (1.06 g.kg-1), AN (20.07 mg.kg-1), C:N (14.63), NH4+-N (10.63 mgkg-1), and NO3--N(11.06 mg.kg-1) than NPK and CK. Such increase in physicochemical properties in partial replacement of inorganic with organic fertilizers plots resulted from higher carbon and nitrogen enzymes activities (BG (143.17 nmol.g-1.h-1), NAG (153.96 nmol.g-1.h-1), and LAP (153.48 nmol.g-1.h-1)) compared to NPK and CK, and further the person correlation and RDA analysis confirmed a significant positive correlation between SOC, AN, and soil enzymes. This study presents a new strategy for assessing the impact of partially replacing inorganic fertilizers with organic fertilizers in rubber plantations, mainly by modifying the soil nutrient composition in tropical regions.

Currently, obesity is a critical global public health burden. Numerous studies have demonstrated the regulation of the pathogenesis of obesity and metabolic abnormalities by the gut microbiota and microbial factors; however, their involvement in the various degrees of obesity is not yet understood well. Previously, obesity has been shown to be associated with decreased levels of vitamin B12. Considering exclusive microbial production of vitamin B12, we hypothesised that a decrease in cobalamin levels in obese individuals may be at least partially caused by its depleted production in the intestinal tract by the commensal microbiota. In the present study, our aim was to estimate the abundance of enzymes and metabolic pathways for vitamin B12 synthesis in the gut microbiota of mouse models of alimentary and genetically determined obesity, to evaluate the contribution of the obesogenic microbiome to vitamin B12 synthesis in the gut. We have defined a significantly lower representation of enzymes and metabolic pathways for vitamin B12 biosynthesis in obese mice compared to non-obese mice, wherein enzyme depletion was more pronounced in lepr(-/-) (db/db) mice, which developed severe obesity. The representation of enzymes involved in cobalamin synthesis is strongly correlated with representation of several microbes in high-fat diet fed mice, whereas there were almost no correlations in db/db mice. Therefore, the degree of obesity and the composition of the correspondent microbiota are the main contributors to the representation of genes and pathways for cobalamin biosynthesis in the mouse gut.

Transketolase (TKT) is an essential thiamine diphosphate (ThDP)-dependent enzyme of the non-oxidative branch of pentose phosphate pathway, with the glucose-6P flux through the pathway regulated in various medically important conditions. Here, we characterize the brain TKT regulation by acylation in rats with perturbed thiamine-dependent metabolism, known to occur in neurodegenerative diseases. The perturbations are modelled by administration of oxythiamine inhibiting ThDP-dependent enzymes in vivo, or by reduced thiamine availability in the presence of metformin and amprolium, inhibiting intracellular thiamine transporters. Compared to control rats, chronic administration of oxythiamine does not significantly change the modification level of the two detected TKT acetylation sites (K6 and K102), but doubles malonylation of TKT K499, concomitantly decreasing 1.7-fold the level of demalonylase sirtuin 5. The inhibitors of thiamine transporters do not change average levels of TKT acylation or sirtuin 5. TKT structures indicate that the acylated residues are distant from the active sites. The acylations-perturbed electrostatic interactions may be involved in conformational shifts and/or formation of TKT complexes with other proteins or nucleic acids. Acetylation of K102 may affect the active site entrance/exit and subunit interactions. Correlation analysis reveals that the action of oxythiamine is characterized by significant negative correlations of K499 malonylation or K6 acetylation with TKT activity, not observed upon the action of the inhibitors of thiamine transport. However, the transport inhibitors induce significant negative correlations between the TKT activity and K102 acetylation or TKT expression, absent in the oxythiamine group. Thus, perturbations in the ThDP-dependent catalysis or thiamine transport manifest in the insult-specific patterns of the brain TKT malonylation and acetylations.

Immobilization of enzymes is a good field of study to expand the life of enzyme in and lowering the cost of the chemical processes such as separation processes. Urease is an important enzyme with medical and industrial applications. The aim of the present study is to prepare an immobilized urease on a strong cation exchange resin (Amberlite IR120 -Na) and study of its activity and stability. We monitored the liberation of Na ions in the collected fractions and searching for protein in the fractions as an indicators of immobilization by ion exchange phenomenon. Sodium is measured using atomic absorption spectroscopy techniques, while protein tested by Bradford’s method. Immobilized urease activity was evaluated by salicylate-hypochlorite method. The results indicated a complete immobilization of urease enzyme on the resin surface with reserving 92% of the activity of free enzyme. The immobilized urease enzyme on resin showed good stability and it has a 62% of its activity after 154 days of storage at room temperature. It is concluded that a new immobilized urease enzyme system is prepared with good enzyme activity and stability.

Extracellular enzymes catalyze biogeochemical reactions in soil, cycling carbon and nutrients in agricultural systems. Enzymes respond quickly to soil management, including organic amendment inputs, such as biochar, a charcoal-like solid byproduct of bioenergy production. In a previous agricultural field trial, a pine biochar amendment caused an approximately 40% decrease in the enzyme activities of β-glucosidase (BG) and phosphatase (PHOS). The large surface area of the pine biochar has the potential to sorb nutrients and other organic molecules. To test if sorption caused decreased enzyme activity, we used a laboratory assay to quantify the activity of two sorbed enzymes: BG and acid PHOS, involved in the cycling of carbon and phosphorous. The enzymes were incubated with three solid phases: (1) the high surface area pine biochar, (2) the agricultural soil, and (3) a low surface area grass biochar, for an additional comparison. We quantified the sorbed enzymes at pH 6, 7, and 8, using a Bradford protein assay, and measured the immobilized enzyme activities via high-throughput fluorometric analysis. After sorption onto pine biochar, detectable BG and PHOS activity levels dropped by over 95% relative to the soil, supporting direct sorption as one mechanism that reduces enzyme activity in biochar amended soil. This laboratory assay demonstrated that sorption could account for the lack of priming of native soil organic matter and changes in soil phosphorous cycling after pine biochar addition.

Soil enzyme activity can be affected by both production and degradation, as enzymes can be degraded by proteases. However, the impact of nutrient addition on enzyme activity is often solely attributed to changes in enzyme production without fully considering degradation. Here, we demonstrated that the activities of β-1,4-glucosidase (BG), β-D-cellobiohydrolase (CBH), β-1,4-Xylosidase (BX), and β-1,4-N-acetyl-glucosaminnidase (NAG) were comparable in nitrogen (N) and phosphorus (P) fertilized soils and the unfertilized control under field conditions, but the reduction in activity was substantially greater in the fertilized soils during short-term laboratory incubation. The results show that the interruption of the natural, continuous supply of organic matter or non-soil microbial-derived enzymes, which typically occurs under field conditions, leads to a more significant reduction in apparent enzyme activities in fertilized soils compared to unfertilized control. This may be attributed to the higher abundance of protease in fertilized soils, resulting in faster enzyme degradation. Interestingly, P fertilization alone did not have a similar effect, indicating that N fertilization is likely the main cause of the larger decreases in enzyme activity during incubation in fertilized soils compared to unfertilized control soils, despite our study site being poor in P and rich in N. These findings highlight the importance of considering enzyme degradation when investigating material dynamics in forest ecosystems, including the impact of nutrient addition on enzyme activity, as enzyme production alone may not fully explain changes in apparent enzyme activity.

Enzymes are in high demand for very diverse biotechnological applications. However, natural biocatalysts often need to be engineered for fine-tuning their properties towards the end applications, such as the activity, selectivity, stability to temperature or co-solvents, and solubility. Computational methods are increasingly used in this task, providing predictions that narrow down the space of possible mutations significantly and can enormously reduce the experimental burden. Many computational tools are available as web-based platforms, making them accessible to non-expert users. These platforms are typically user-friendly, contain walk-throughs, and do not require deep expertise and installations. Here we describe some of the most recent outstanding web-tools for enzyme engineering and formulate future perspectives in this field.

There is limited data on pregnancy outcomes in Pompe Disease (PD) resulting from deficiency of the lysosomal enzyme acid alpha-glucosidase. Late-onset PD is characterized by progressive proximal muscle weakness and decline of respiratory function secondary to the involvement of the respiratory muscles. In a cohort of twenty-five females, the effects of both PD on the course of pregnancy and the effects of pregnancy on PD were investigated. Reproductive history, course of pregnancy, use of Enzyme replacement therapy (ERT), PD symptoms, and outcomes of each pregnancy were obtained through a questionnaire. Among 20 subjects that reported one or more pregnancies, one subject conceived while on ERT and continued therapy through two normal pregnancies with worsening of weakness during pregnancy and improvement postpartum. While fertility was not affected, pregnancy may worsen symptoms, or cause initial symptoms to arise. Complications with pregnancy or birth were not higher, except for an increase in the rate of stillbirths (3.8% compared to the national average of 0.2-0.7%). Given small sample size and possible bias of respondents being only women who have been pregnant, further data may be needed to better analyze the effects of pregnancy on PD, and the effects of ERT on pregnancy outcomes.

In bacteria, enzymes usually comprise of enzymatic domains only. In eukaryotes/mammals, and here particularly in context of signal transduction, proteins with enzymatic functions contain additional modules promoting interactions with cooperating signaling proteins. Often such protein-protein interaction domains, such as SH2- and SH3-domains, help to localize the enzymatic function (e.g. kinase, phosphatase) to another protein, cellular compartment, or membrane nanodomain. In such cases, the interaction domain(s) serve(s) the enzymatic function. However, there are molecular situations in which an interaction domain fulfills its task completely independent of the protein´s enzymatic function and in this review, we call such function the adapter function of an enzyme. Though many stories can be told, we will concentrate on several proteins playing crucial roles in cells of the immune system, such as BTK, PI3K, and SHIP1, as well as in cancer cells, such as proteins of the RAS/ERK MAPK pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors, thereby highlighting the need to develop pharmacological approaches, such as PROTACs, that eliminate the entire protein. We also review how genetic kock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

The catalytic parameters describing enzyme catalysis are discussed in the context of the design of de novo enzymes. It is shown that high values of the catalytic efficiency may in some cases reflect tight substrate binding and lead to an immediate levelling-off of rates to reach a low plateau, an undesirable scenario for many industrial applications. It is argued that increasing the catalytic rate constant should be a main target in de novo enzyme engineering.

Determining halal product of fermented foods and bevearages becomes important for muslim consumers due to religious codification of halal. In order to help on site analysis, an alcohol biosensor was under development. The biosensor was constructed using bacteria producing alcohol oxidase (AOX). Bacteria from apple juice was isolated in advance to obtain pure bacteria. The bacteria was cultured in two solid media. Bacteria on the Acetobacter media had a resistance in a solution containing alcohol with a higher concentration. Acetobacter was cultured on solid and liquid media for measuring at various ethanol concentrations (0.01%-3%) with different absorbance value. Bacteria with absorbance value 0.500 had the highest oxidation current peak. Later, it was used as bioreceptor for measuring ethanol by cyclic voltammetry method. The linearity of measurement later was compared with spectrophotometric measurement. By using cyclic voltammetry the linearity had higher R² value than spectrophotometric method. However, it only had stability for two days, there fore it needs more improvement for lasting its lifetime. Basically, electrochemical method has potency for further being improved fur as an alternative method of ethanol on-site measurement.

Wolfenden and his coworkers observed the astronomical numbers for the catalytic proficiency of some enzymes. We connected that pinnacle of biological evolution to the universal thermodynamic evolution. We added or multiplied a random noise with chosen rate constants to explore the correlation between dissipation and enzyme efficiency for ten enzymes: beta-galactosidase, glucose isomerase, β-lactamases from three bacterial strains, ketosteroid isomerase, triosephosphate isomerase, and carbonic anhydrase I, II, and T200H. The turnover number kcat and catalytic efficiency kcat/KM are proportional to the overall entropy production – the main parameter from irreversible thermodynamics. For most enzymes with the Michaelis-Menten type cycle kinetics, the best increase in the forward kcat/KM follows after increasing the equilibrium constant of substrate-enzyme association. The Discussion section emphasizes the role of biological evolution in harvesting order (high enzyme efficiency) from disorder (high noise and dissipation). It also connects the applications of the maximum partial entropy production theorem in optimizing enzyme kinetics (D. Juretić "Bioenergetics - A Bridge across Life and Universe") with the present total entropy production role analysis. De novo enzyme design and various attempts to speed up the rate-limiting catalytic steps may profit from our theoretical insights.

Electrochemical biosensors based on immobilized enzymes are among the most popular and commercially successful biosensors. Literature in this field suggests that modification of electrodes with nanomaterials is an excellent method for enzyme immobilization, which can greatly improve the stability and sensitivity of the sensor. However, the poor stability, weak reproducibility, and limited lifetime of the enzyme itself still limit the requirements for the development of enzyme electrochemical biosensors for food production process monitoring. Therefore, constructing sensing technologies based on enzyme electrochemical biosensors remains a great challenge. This article outlines the construction principles of four generations of enzyme electrochemical biosensors and discusses the applications of single-enzyme systems, multi-enzyme systems and nano-enzyme systems developed based on these principles. The article further describes methods to improve enzyme immobilisation by combining different types of nanomaterials such as metals and their oxides, graphene-related materials, metal-organic frameworks, carbon nanotubes and conducting polymers. In addition, the article highlights the challenges and future trends of enzyme electrochemical biosensors, providing theoretical support and future perspectives for further research and development of high-performance enzyme chemical biosensors.

Background: Bacterial infections constantly have a large impact on public health, because of increased rates of resistance and reduced frequency of development of novel antibiotics. The utility of conventional antibiotics for treating bacterial infections has become increasingly challenging. The aim of the study was to assess the antibacterial effect of β -Lapachone, a novel synthetic compound. Methods: The antibacterial activity of the β -Lapachone compound was examined against laboratory strains by agar well diffusion method, minimal inhibitory concentration (MICs), and minimal bactericidal concentration (MBCs). Growth kinetics inhibition in presence of β -Lapachone on Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa (ATCC 27853) was assessed by MABA. Crystal violet blue assay was used for biofilm inhibition assays, biofilm eradication assay and for molecular modeling PyMOL was used. Results: β -Lapachone exhibited potent antimicrobial activity against laboratory strains of bacteria with MIC of 0.2 mM for S. saprophyticus and Staphylococcus aureus, and 0.04 mM for Staphylococcus epidermidis and Pseudomonas aeruginosa ATCC 27853. The inhibition of catalase enzyme was found to be the cause for its antibacterial activity. Molecular modeling predicted the binding of β -Lap at active site and heme binding site of catalase, KatA. The activity of some commercial antibiotics was enhanced in association with β -Lap. In addition, β -Lap inhibited the biofilm formation and eradicated the already formed and ultra-mature biofilms of aforesaid bacterial strains.

Genome-scale metabolic model (GEM) is a powerful tool for interpreting and predicting cellular phenotypes under various environmental and genetic perturbations. However, GEM only consid-ers stoichiometric constraints, and the simulated growth and product yield values will show a monotonic linear increase with increasing substrate uptake rate, which deviates from the experi-mentally measured values. Recently, the integration of enzymatic constraints into stoichiometry-based GEMs was proven to be effective in making novel discoveries and predicting new engineer-ing targets. Here we present the first genome-scale enzyme-constrained model (eciCW773) for Corynebacterium glutamicum reconstructed by integrating enzyme kinetic data from various sources using ECMpy workflow based on the high-quality GEM of C. glutamicum (obtained by modifying the iCW773 model). The enzyme-constrained model improved the prediction of pheno-types and simulated overflow metabolism, while also recapitulating the trade-off between biomass yield and enzyme usage efficiency. Finally, we used eciCW773 to identify several gene modifica-tion targets for L-lysine production, most of which agree with previously reported genes. This study shows that incorporating enzyme kinetic information into the GEM enhances the cellular phenotypes prediction of C. glutamicum, which can help identify key enzymes and thus provide reliable guidance for metabolic engineering.

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses, cellular protein homeostasis. Translation of full-length (Met1) IDE transcripts has reported to result in targeting to mitochondria but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, here we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that mitochondrial IDE interacts strongly with mitochondrial ribosomes as well as with proteins of the respiratory chain. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any proteolytic IDE substrates. We speculate that IDE could adopt similar functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses.

In order to further clarify the growth-promoting effect of non-core Metarhizium spp. M. marquandii on plants, M. marquandii SGSF043, which was obtained from the pre-screening in the laboratory, was selected as a test strain, and the effects of the fungus fermentation broth during the stabilization period of the growth of this strain on the growth and growth of maize seeds were investigated by using fermentation broth as a submerged seed. germination, seedling growth, and inter-root soil enzyme activity of seedlings. The results are as follows: In the seed germination test, M. marquandii SGSF043 fermentation liquid had a certain inhibitory effect on the germination of maize seeds, and the 10-times dilution of the fermentation liquid significantly promoted the seed germination, and the effect was the best; After the germination test, the growth of maize germ was promoted by the treatment groups of 10 times and 1,000 times diluent; In the field plot experiment, the 100-fold dilution had the best growth promotion effect, which could promote the growth of cabbage seedlings and increase the seedling biomass. The results show that M. marquandii SGSF043 has the potential to promote the growth of maize and improve the soil environment, which provides a theoretical basis for the research and application of M. marquandii in farmland.

The primary contributor to global warming has been the careless usage of fossil fuels. Urbanization's threat to the depletion of these resources has made it necessary to find alternatives due to the rising demand. Four different forms of biofuels are now available and constitute a possible replacement for fossil fuels. The first generation of biofuels is generated from the edible portion of biomass, the second generation is made from the non-edible portion of biomass, the third generation is made from algal biomass, and the fourth generation is made using molecular biology to improve the algal strain. Second generation biofuels are extremely important because they are derived from non-edible biomass, such as agricultural and agro-industrial wastes rich in cellulose, hemicellulose, pectin, and starch impregnated with lignin, and are hydrolyzed after delignification by physio-chemical or biological pretreatments using ligninases. There has been a lot of research on their production using chemical and enzymatic hydrolysis, but it has not been economically viable in comparison to first generation biofuels due to the formation of several inhibitors with chemical hydrolysis and the high cost of the enzymes. Furthermore, the need for multiple enzymes due to the various types of carbohydrates in the feedstocks makes the enzymatic process too expensive. This article examines the enzymes involved in the hydrolysis of feedstocks for the production of second generation bioethanol, a highly acceptable biofuel.

Some cancer cells rely heavily on non-essential biomolecules for survival, growth, and proliferation. Enzyme based therapeutics can eliminate these biomolecules, thus specifically targeting neoplastic cells; however, enzyme therapeutics are susceptible to immune clearance, exhibit short half-lives, and require frequent administration. Encapsulation of therapeutic cargo within biocompatible and biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) is a strategy for controlled release. Unfortunately, PLGA NPs exhibit burst release of cargo shortly after delivery or upon introduction to aqueous environments where they decompose via hydrolysis. Here we show the generation of hybrid silica-coated PLGA (SiLGA) NPs as viable drug delivery vehicles exhibiting sub-200 nm diameters, a metastable Zeta potential, and high loading efficiency and content. Compared to uncoated PLGA NPs, SiLGA NPs offer greater retention of enzymatic activity and slow the burst release of cargo. Thus, SiLGA encapsulation of therapeutic enzymes, such as asparaginase, could reduce frequency of administration, increase half-life, and improve efficacy for patients with a range of diseases.

In light of the hazardous effects of global warming brought on by climate change, bioethanol production is one of the key alternatives in which the world has expressed a great deal of interest. Two factors—indigenous production and price—are crucial for bioethanol to be appealing in Africa and, for that matter, Ghana. The objective of this research is to produce bioethanol from cheap lignocellulosic materials using onsite enzymes produced from cassava peels degrading fungi. Due to Ghana's prominent position as one of the leading cassava producers in the world, the study focused on utilizing cassava peels, which are underutilized in the country. The research was conducted in multiple stages. In the first stage, the peels were prepared by washing, drying, and manually crushing them using mortar and pestle. The second stage involved analyzing the chemical composition of the peels, including starch and insoluble fibre, which were determined as Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), and Acid Detergent Lignin (ADL). The results showed significant levels of cellulose (39.78%) and starch (31.21%), indicating that cassava peels are valuable raw materials for bioethanol production. The content of hemicellulose (21.11%) and lignin (3.84%) were also determined. In the third stage, using a Petri dish, three fungi (ICPF1, ICPF2, and ICPF3) were isolated from two different cassava peels. ICPF1 was only identified from fresh cassava peels (FCP), while all three fungi were identified from decayed cassava peels (DCP). Morphologically, these fungi were identified as Aspergillus niger, Aspergillus flavus, and Rhizopus stolonifer. The fourth stage focused on optimizing the enzyme activity of the three isolates for potential applications, with A. niger demonstrating the highest enzyme activity with a diameter of zone of clearance of 16 mm. Stage five involved optimizing the production of the onsite enzyme in a 50ml flask using A. niger, basal salt medium (BSM), and cassava peels as a substrate. The DNSA method was used to measure the absorbance of maltose and glucose at 540 nm for various substrate concentrations (1%, 3%, 5%, 8%, and 10%) at specific intervals of 2, 4, 6, 8, and 10 days with a spore concentration of 2.1 x 105 cells/ml. The maltose and glucose concentrations were calculated as 7.138mg/ml and 6.398 mg/ml, respectively, and the corresponding enzyme activity was determined as 4.759U/ml and 4.265U/ml. The optimal conditions of Day 4 and a substrate concentration of 10%, along with a fixed temperature of 30°C and a pH of 5.5, were used to prepare the onsite enzymes in a 500 ml flask for the fermentation process. The onsite enzymes were used for saccharification and Saccharomyces cerevisiae for fermentation under simultaneous saccharification and fermentation (SSF) process. A mixture of 20 ml of onsite enzymes and 1.5 g of S. cerevisiae were added to substrate concentrations of 5%, 10%, and 20%, and the ethanol concentration was analyzed daily for 7 days using Gas Chromatography (GC). The highest ethanol concentration (1.316%) was observed on Day 5 with a substrate concentration of 20%, while the lowest concentration (0.123%) was recorded on Day 1 with a substrate concentration of 5%.

A recent report from the American Heart Association in 2018 shows that over 103 million American adults have hypertension. The angiotensin-converting enzyme (ACE) (EC 3.4.15.1) is a dipeptidyl carboxylase that, when inhibited, can reduce blood pressure through the renin-angiotensin system. ACE inhibitors are used as a first-line medication to be prescribed to treat hypertension, chronic kidney disease, heart failure among others. It has been suggested that ACE inhibitors can reduce the symptoms in mouse models. Despite the benefits of ACE inhibitors, previous studies also have suggested that alterations in the ACE gene are risk factors for Alzheimer’s disease (AD) and other neurological diseases. In mice, overexpression of ACE in the brain reduces symptoms of the AD-model systems. Thus, we find opposing effects of ACE on health. To clarify the effects, we dissect the functions of ACE as follows: (1) angiotensin-converting enzyme that hydrolyzes angiotensin I to make angiotensin II in the renin-angiotensin system; (2) amyloid-degrading enzyme that can hydrolyze beta-amyloid and reduce amyloid toxicity. The efficacy of the ACE inhibitors is well established in humans, while the knowledge specific to AD remains to be open for further research. We provide an overview of ACE and inhibitors that link a wide variety of age-related comorbidities from hypertension to Alzheimer’s disease to aging. ACE also serves as an example of the middle-life crisis theory that assumes deleterious events during the midlife, leading to age-related later events.

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.

Prosthetic infections are associated with high morbidity, mortality, and relapse rates making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, the work proposed a method of combating pathogenic microorganisms by modifying the surface of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors - 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on immobilization of biologically active molecules, it is important to monitor changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g. FTIR) are ideal. The ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surface of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride was demonstrated.

Catalase can decompose hydrogen peroxide in plants under controlled and stressed condi-tions. In a stressed environment, the increase in hydrogen peroxide production makes catalase activity a major defense system for plant growth. The current study was conducted to evaluate the catalase activity of pre-isolated and identified bacterial strains Bacillus aryabhattai (AN30), Bacillus megaterium (AN24), Bacillus megaterium (AN31) and Bacillus megaterium (AN35) and their potential for rice seedlings growth promotion. These strains were characterized for quantita-tive catalase, urease, siderophores, and exopolysaccharides production using LB media. Sub-sequently, the effectiveness of these strains was checked by quantifying the catalase activity in the rhizosphere of rice seedlings. Furthermore, the catalase activity was quantified in the roots and shoots of rice seedlings. Plant growth parameters were also studied in a growth room trial. The strain AN24 showed the maximum catalase activity (1.36 mol cm-1), urease ac-tivity (1.35 mol cm 1), exopolysaccharides (4.20 µg mL-1), and siderophores (2.32%) production in LB media. All tested strains showed significantly higher catalase activity in soil compared to control. Among sole application, strain AN24 showed better results; however, consortium application of strains AN24+AN30+AN35+AN31 showed the maximum increase in catalase ac-tivity in the rhizosphere of rice seedlings. Moreover, consortium application of AN24+AN30+AN31+AN35 resulted in the highest increase in catalase activity, dry biomass of shoots and roots, shoot and root length of rice seedlings. The results showed that a consortium of these Bacillus strains with catalase activity has greater potential to enhance an antioxidant defense system and growth promotion of rice seedlings. However, further experimentation under natural conditions is required before using these strains as potential bioinoculants for improving the rice growth and yield.

The intensive global use of glyphosate has led to the evolution of glyphosate resistant (GR) weed species, including the economically damaging horseweed (Conyza sumatrensis). We evaluated the glyphosate resistance mechanisms of C. sumatrensis. While 5-enolpyruvylshikimate-3-phosphate synthase activity was similar between the glyphosate resistant (GR) and nonresistant biotypes, plants from the GR population accumulated lower shikimate levels than susceptible ones, suggesting the absence of target-site resistance mechanisms. Decreases over time in glyphosate concentrations in GR leaves were not accompanied by increases in glyphosate concentrations in their stem and roots, indicating lower glyphosate distribution rates in GR plants. The early appearance of aminomethylphosphonic acid (the main glyphosate metabolite) in leaves, as well as its presence only in the stems and roots of GR plants, suggests faster glyphosate metabolism in GR plants than in susceptible ones. GR plants treated with glyphosate also showed greater antioxidant (ascorbate peroxidase [APX] and catalase [CAT]) and cytochrome P450-enzyme activities, indicating their great capacity to avoid glyphosate-induced oxidative stress. Three non-target mechanisms (reduced glyphosate translocation, increased metabolism, and increased antioxidant activity) therefore confer glyphosate resistance in C. sumatrensis plants. This is the first time that APX, CAT and P450 enzyme activities are related to GR in C. sumatrensis.

Rapid industrialization, urbanization, and population explosion in sub-Saharan Africa escalate environmental Lead levels with subsequent elevation of blood Lead levels in children. Nutrition status, age, and genetics govern one’s susceptibility to Lead toxicity. This study expounded this susceptibility by relating blood Lead levels, d-aminolevulinic acid dehydratase enzyme activity (ALAD), and genetic variations of proteins that code for ALAD enzyme in urban children of Uganda. Spectrophotometric analysis for blood Lead (BL), hemoglobin levels, and determination d-levels aminolevulinic acid dehydratase enzyme activity of the blood samples from 198 children were performed prior to a polymerase chain reaction and restriction fragment length digestion for ALAD polymorphism was done. Up to 99.5% of samples coded for the ALAD1 allele whereas 0.05% coded for ALAD2. Genotypes ALAD2-2 members had elevated BLL (mean 14.1 µg/dL) and reduced ALAD enzyme activity compared to others. This, therefore, implies that the majority of children hoard BL which may affect them later in life.

Current paper reviews applications of luminescence bioassays for monitoring low-intensity factors, namely, radioactivity of different types (alpha, beta and gamma), and bioactive compounds (humic substances and fullerenols). Luminescence intensity is taken as a physiological parameter of luminous organisms. High rates of luminescence response can provide (1) a proper number of experiments under comparable conditions and, therefore, proper statistical processing, with this being highly important for ‘noisy’ low-dose exposures; (2) non-genetic, i.e. biochemical and physicochemical mechanisms of cellular response, in accordance to “exposome” concept. Bioassays based on luminous marine bacteria, their enzymes, and fluorescence coelenteramide-containing proteins were used to compare results of low-intensity exposures at cellular, biochemical and physicochemical levels, respectively. Results of the cellular exposures were discussed in terms of hormesis concept. Bioluminescence time dependence under low-dose radiation exposures corresponded to hormesis or threshold models; no bioluminescence monotonic dependency on intensity of exposure (dose rate, radioactivity, concentration) was found. Bioluminescence activation and absence of its dependency on intensity of exposure can be accepted as features of cellular adaptive response. Changes of biological luminescence were analyzed and discussed for bioassays of lower organization level – enzymes and florescent protein.

An in vitro medium-throughput screen using M. tuberculosis H37Rv was employed to screen an in-house library of structurally diverse compounds for antimycobacterial activity. From this initial screen, eleven 7-substituted coumarin derivatives with confirmed monoamine oxidase-B and cholinesterase inhibitory activities, demonstrated growth inhibition of more than 50% at a 50 µM concentration. This prompted further exploration of all the 7-substituted coumarins in our library, nineteen in total, as potential antimycobacterial agents. Four derivatives showed promising antimycobacterial activity with MIC₉₉ values of 8.31 – 29.70 µM and 44.15 – 57.17 µM on M. tuberculosis H37Rv in independent assays using Gaste-Fe and 7H9 + OADC media, respectively. These compounds were found to bind to albumin which may explain the variations in MIC between the two assays. Preliminary antimycobacterial evaluation of moxifloxacin resistant M. tuberculosis show that these compounds are able to maintain their activity in fluoroquinolone resistant mycobacteria. Analysis of structure activity relationships for antimycobacterial versus neuronal enzyme inhibitory activity indicate that structural modification on position 4 and/or 7 of the coumarin scaffold may be utilized to improve selectivity towards either inhibition of neuronal enzymes or antimycobacterial effect. Cytotoxicity evaluations of the compounds indicate moderate cytotoxicity with slight selectivity towards mycobacteria. Further neuroprotective assays on SH-SY5Y human neuroblastoma cells indicate significant neuroprotection for selected compounds irrespective of their neuronal enzyme inhibitory properties. These coumarin molecules are thus interesting lead compounds that may provide insight into the design of new antimicrobacterial and/or neuroprotective agents.

This study reports the preparation of silica and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. Catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated with Michaelis-Menten equation and Line-weaver Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and temperature of 35 ℃. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated by an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Thrombosis is characterized by the pathological formation of fibrin clots within a blood vessel, leading to the obstruction of blood flow. Fibrinolytic enzymes from microorganisms have exhibited promising effects to dissolve clots in a more efficient and safe way. Then, the aim of this study was to evaluate the biomass and fibrinolytic enzyme production of Tetradesmus obliquus under autotrophic and mixotrophic conditions using different concentration of corn steep liquor (CSL). Different extraction and precipitating methods were tested, and the enzyme was purified by ion exchange chromatography. More advantageous culture condition was mixotrophic using 0.25% CSL, showing the highest values of biomass productivity (Px = 169.3 ± 44.36 mg∙L-1day-1) and specific growth rate (µmax = 0.17 ± 0.00 day-1), and significant fibrinolytic production (391.34 ± 40.03 U∙mg-1). Moreover, fibrinolytic activity was higher when extracted by homogenization and precipitated using acetone, which exhibited clear zone of fibrin degradation in the fibrinolytic plate assay. Additionally, the purified enzyme showed specific activity of 1176.90 ± 140.37 U∙mg-1 and molecular weight around 97 kDa. Finally, the enzyme has higher enzymatic activity than various fibrinolytic enzymes, and the obtained enzyme has potential to be developed as a therapeutic agent in thrombosis treatment. Additional studies are need to investigate the biochemical properties and biological profile of this enzyme.

Various peptides or non-structural amino acids are recognized by their specific target proteins and perform biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein-peptide complexes. To obtain structural information on substrate recognition of peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as main-chain of peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of main-chain. One of the triglycine-binding conformations was identical with the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application X-ray crystallography to identify substrate recognition sites in peptide binding enzymes.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wildtype subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers by a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as target, yielding synthetic multienzyme-complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was achieved also on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable, TVCVPA-assisted electrochemical biosensing through modularly IgG-assembled sensor enzymes.

The TGACG motif-binding factor1 (TGA1) transcription factor, a significant protein in the bZIP transcription factor family, exhibits a myriad of functions, contributes to numerous biological events, and holds substantial application potential. In this study, our analysis revealed a diverse range of photoregulatory and hormone regulatory elements within the MtTGA1 promoter region. The expression profile of MtTGA1 indicated its highest expression in the root, with its regulation influenced by SA, ABA, BR, and GA. Under salt stress conditions, transgenic plants demonstrated significantly longer root lengths and heightened activities of antioxidant enzymes such as ascorbic acid catalase (APX), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in their roots and leaves compared to control plants. Simultaneously, the levels of endogenous hormones ABA and BR escalated in transgenic plants, with a marked change in the morphology of their leaf cells. Transcriptome analysis identified a total of 193 differentially expressed genes, implicating a wide array of biological processes. Furthermore, we found that MtTGA1 is able to interact with Medicago truncatula SPX domain containing protein 1 (SPX1). In conclusion, the MtTGA1 transcription factor plays a crucial role in enhancing salt tolerance, which provides new insights for improving plant survival under salt stress conditions.

The lab-on-a-chip concept, enabled by microfluidic technology, promises the integration of multi-ple discrete laboratory techniques into a miniaturised system. Research into microfluidics has generally focused on the development of individual elements of the total system (often with rela-tively limited functionality), without full consideration for integration into a complete fully opti-mised and miniaturised system. Typically, the operation of many of the reported lab-on-a-chip devices is dependent on the support of a laboratory framework. In this paper, a demonstrator platform for routine laboratory analysis is designed and built, which fully integrates a number of technologies into a single device with multiple domains such as fluidics, electronics, pneumatics, hydraulics, and photonics. This facilitates the delivery of breakthroughs in research, by incorpo-rating all physical requirements into a single device. To highlight this proposed approach, this demonstrator microsystem acts as a fully integrated biochemical assay reaction system. The re-sulting design determines enzyme kinetics in an automated process and combines reservoirs, three-dimensional fluidic channels, optical sensing, and electronics in a low-cost, low-power and portable package.

Prevention practices have been extensively used to contain the spread of the SARS-CoV-2 virus. These include social distancing, wearing masks, disinfection of hands, and sanitization of contact surfaces. However, the excessive usage of chemical disinfectants pose long term adverse effects to human health and the environment. Development of effective and environmentally friendly biocides, or virucidal agents, will help mitigate the ill effects of chemical disinfectants. Enzymes are potential candidates for the preparation of biocides against bacteria and viruses. Exploration of the virucidal activity of commercial enzymes, will highlight prospective, readily available sources for research on enzyme based biocides. In this study, the virucidal effect of some com-mercial enzyme preparations has been investigated against the SARS-CoV-2 virus. Vida Defense (2000 µg/ml), Excellacor (1500 µg/ml), and SEBkinase (3000 µg/ml) reduced SARS-CoV-2 viral ti-ters by ≥1 log CCID50 (≥90%). ImmunoSEB (6000µg/ml) and Peptizyme SP (500µg/ml) reduced the SARS-CoV-2 viral titers by 0.8 log CCID50 (84.2%). The study indicates that enzyme prepara-tions offer the potential to be explored further for an anti-viral biocide against SARS‐CoV‐2 for reducing the risk of COVID‐19 transmission. However, further studies are mandated to improve efficacy and establish safety.

The outbreak across the globe due to coronavirus disease 2019 (COVID-19) has spread abruptly by infected humans worldwide. The continuous efforts by scientists is on way to understand how pandemic of COVID-19 resembles and differs from serve acute respiratory syndrome coronavirus (SARS-CoV) at transcriptomic and genomic level. The SARS-CoV and COVID-19 exploits the angiotensin converting enzyme 2 (ACE2) receptor to gain entry inside the cells.We analyzed the entry COVID-19 into host cell due to receptor binding domain (RBD) of spike glycoprotein. The proposed simulation data shows similar ternary structures from two viruses shares approximately 80 percent identity in amino acid sequences. Our molecular modeling investigation signifies that angiotensin converting enzyme 2 (ACE2) has stronger interaction with COVID -19 RBD. The Amino acid phenylaniline F486 LOOP plays vital role due to its penetration into hydrophobic pocket in ACE2.The said investigation of S-Glycoprotein RBD of COVID-19 or SARS-CoV-2 via ACE2 provides post genome analysis of protein-protein interaction for rapid assessing transmission of infected patients by deadly CoVID-19. The scientific data extracted implies early guidance to control and viral prevention of CoVID-19.

Recently, the outbreak of coronavirus disease 2019 (COVID-19) is threatening human health globally. There is a dire need to find potential therapeutic agents. Angiotensin converting enzyme 2 (ACE2), as an entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered as potential therapeutic target in COVID-19 pandemic. Here, our bioinformatics analysis revealed that the biological function of ACE2 was correlated with regulation of blood pressure and mediation of SARS-CoV-2 entry into host cells. Ten ACE2 cooperative proteins were identified by using STRING with a high score. ACE2 expressed highly in the small intestine, testis, and kidney. The level of ACE2 expression in tumor tissues varies in different types of cancers compared with that in normal tissues. It was worth noting that the expression level of ACE2 in the tumor has no effect on patient survival. MiRNA hsa-miR-942-5p, and three transcription factors (TFs) including Signal transducer and activator of transcription 4 (STAT4), Estrogen related receptor α (ESRRA), and Signal transducer and activator of transcription 3 (STAT3) were selected as novel ACE2 regulators. Moreover, nine potential therapeutic drugs were predicted by two online databases. Thus, our research may expand the overall view of ACE2 in COVID-19 treatment.

Background: Catalase is a vital antioxidant enzyme that dismutates H₂O₂ into water and molecular oxygen. Many protocols have been developed to measure catalase enzyme activity. Spectrophotometric methods are the most common assays that used to assess catalase enzyme activity. Methods: Because the rate-limiting step during catalase enzyme activity depends upon the dissociation of hydrogen peroxide, the developed assay measures the reaction between a hydroquinone/anilinium sulfate/ammonium molybdate reagent and Unreacted Hydrogen Peroxide, which results in the production of a purple, disubstituted quinone compound with a maximum absorbance value at 550 nm. Results: To clarify the precision of the developed method, the coefficients of variation were determined to be 2.6% and 4.7% for within run measurements and between run measurements, respectively. This method returned results that correlated well (r = 0.9982) with the results returned using the peroxovanadate method to assess catalase enzyme activity. Additionally, we examined the use of the newly developed hydroquinone assay to measure catalase enzyme activity in liver and bacterial homogenate samples. Conclusion: These results demonstrated that this assay can be used for scientific research and routine health applications because it is inexpensive, simple, accurate, and rapid. This method is suitable for use in clinical pathology laboratories because it is simple and produces precise and reproducible results.

The importance of enzymes in the poultry industry is ever increasing because they help to extract as many nutrients as possible from the raw material available and reduce environmental impacts. Therefore, an experiment was conducted to examine the effect of a natural enzyme complex (ASC) on diets low in AME, Ca and P. Ross 308 broilers (n = 900) were fed one of four diets: (1) positive control (PC) with no enzyme added (AME 12.55 MJ/kg, AVPhos 4.8 g/kg and AVCal 9.6 g/kg); (2) negative control (NC) with no enzyme added and down-specified for AME, Ca and P (AME 12.18 MJ/kg, AVPhos 3.3 g/kg, AVCal 8.1 g/kg); (3) negative control plus ASC at 200 g/t; and (4) negative control plus ASC at 400 g/t. Broiler performance, digesta viscosity, tibia mineralization and mineral content were analyzed at d 21. Between d 18 and 20, excreted DM, GE, total nitrogen, Ca, and P were analyzed. ASC at 200 g/t and 400 g/t improved the FCR (p = 0.0014) significantly when compared with that of the NC. There were no significant differences in BW or FI between the treatments. Birds fed ASC at 200 g/t and 400 g/t had significantly improved digesta viscosity (p < 0.0001) compared with that of the PC and NC and had significantly higher excreted DM digestibility (p < 0.01) than the NC and the PC with 400 g/t ASC. ASC inclusion significantly improved P retention (p < 0.0001) compared with that in the PC. Ca retention was significantly increased by 400 g/t ASC compared with that in the PC and NC (p < 0.001). AME was significantly higher (p < 0.0001) for all treatments compared with that in the NC. There were no significant differences between treatments for any of the bone measurements. This study showed that feeding ASC can support the performance of broilers when fed a specification reduced in Ca, P and AME, with the greatest results being seen with the higher inclusion level of ASC.

The production of oil by thraustochytrids is still costly because of its necessary and expensive culture medium. We evaluated the growth, lipid production, and fatty acid composition in an inexpensive, molasses-based (M) medium. Newly isolated thraustochytrid strains were identified as members of genus Aurantiochytrium based on 18S rRNA gene sequences. Isolated strains were repeatedly cultured in M medium for acclimatization to the medium. Acclimatized and non-acclimatized strains were cultured in basal GY medium and M medium. The lipid in the cultured cells were extracted by the Folch method, and the fatty acid composition was analyzed by gas chromatograph. The growth and lipid production in the non-acclimatized strains were significantly inhibited by culturing in M medium, whereas the growth and lipid production were improved by acclimatization treatment in the same medium. The fatty acid composition in the cultured cells were affected by the M medium compared to the basal GY medium. The production of some enzymes such as leucine and valine aryl-amidase was suppressed in the M medium, and the activities recovered by acclimatization treatment. The growth of some strains in the molasses medium was thus recovered by the acclimatization process, and the profiles of extracellular enzymes were also affected.

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. More the evolution of a character is probable more convergent evolution will be witnessed, consequently, convergent evolution could mean that the convergent character evolution result as a combination of these two factors. We investigate this phenomenon by studying the convergent evolution of biochemical functions. We use for the investigation the case of β-lactamases. β-lactamases hydrolyzes β-lactams which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. β-lactamase activity is present in two different superfamilies: the metallo-β-lactamase and the serine β-lactamase superfamily. The mechanism used to hydrolyze the β-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further show that the β-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explaining the promiscuous β-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part, by positive selection as the organisms having β-lactamase activity allows them to resist to organism secreting β-lactams. Indeed a mutation increasing the β-lactamases activity will be selected as the organisms having this activity will have an advantage over the others.

Amylin, also known as islet amyloid polypeptide (IAPP), is a metabolic homeostasis-related hormone that is produced and released by the β cells of the pancreas, the same cells that produce insulin. Insulin-degrading enzyme (IDE) is a protease enzyme that plays an essential role in the breakdown and degradation of various peptides, including insulin and amylin. Direct binding & interaction between amylin and IDE is inextricably linked to the degradation of amylin, and research and development effort in this area is crucial to understand the potential therapeutic implications of disrupting the IDE-amylin interaction in the context of conditions where metabolic homeostasis needs to be regulated, such as diabetes and obesity. Here, this article incorporates currently available experimental complex structure of amylin and IDE, and delves deep into the interstructural biophysics underlying the binding interface of the two interacting partners. With a set of comprehensive structural biophysical analysis, this article identified an intriguing region of high electrostatic potential indicative of strong binding sites between the first N-terminal lysine (Lys1, K1) residue of amylin and Glu341 (E341) of IDE. This unique electrostatic hotspot presented herein paves the way for the rational design of drug-like small molecules that can selectively disrupt this interaction, offering a targeted therapeutic strategy for improved metabolic homeostasis, particularly for patients with diabetes and obesity.

Biochars are often proposed as a strategy for long-term carbon sequestration. Nevertheless, application of pyrolysed feedstock, particularly along with exogenous organic matter, may affect carbon dynamics in soil through introduction of labile carbon pools and stimulation of extracellular enzymes activity. The main aim of this research was to evaluate the influence of biochars and unprocessed organic amendments addition in two agricultural soils on the dissolved organic carbon (DOC) content and activity of three enzymes involved in carbon turnover. In the incubation experiment, activity of dehydrogenase, β-glucosidase, cellulase and DOC content were measured on day 30, 60, 90, 180 and 360. Addition of biochars stimulated the activity of dehydrogenase and β-glucosidase, while cellulase was suppressed. Fresh biomass amendment enhanced activity of the enzymes through priming effect. DOC content tended to be the highest in treatments with high enzyme activity, suggesting that DOC introduced with amendments acted as a source of energy for microbes. Our findings support the hypothesis that biochar properties and presence of exogenous organic matter affect microbial response in soil, what might be crucial for carbon sequestration potential of biochar. However, long-term studies are recommended to fully understand the mechanisms that determine response of soil biota to biochar addition.

Angiotensin-converting enzyme (ACE) inhibitors are used primarily in the treatment of hypertension, heart failure, and in the acute phase of myocardial infarction. Lisinopril [N2-[(1S)-1-car-boxy-3-phenylpropyl]-L-lysyl-L-proline], enalapril [(S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline] and ramipril [2-aza-bicyclo-[3.3.0]-octane-3-carboxylic acid] are all five-membered heterocycles and three of the most prevalent ACE inhibitors in clinical use worldwide. ACEi-induced angioedema (ACEi-AE) is clinically characterized by self-limited edema of the dermis and subcutaneous lipid tissue, localized on face skin, oral mucosa and tongue in most cases. However, severe episodes of intestinal AE misdiagnosed as acute appendicitis and laryngeal AE requiring incubation have been reported. The pathophysiology of ACEi induced-AE is attributed to the accumulation of bradykinin, which is a potent vasodilator with proinflammatory activity that is normally degraded by angiotensin-converting enzyme (ACE) and aminopeptidase P; however, a small proportion of treated patients is affected. Given that patients do not respond to anti-H1 antihistamines and steroids, the early clinical recognition and the ACEi discontinuation are the treatment of choice for the long term management of ACEi-AE.

Oxidative stress is defined as an imbalance between the production of free radicals and reactive oxygen species (ROS) and the ability of the body to neutralize them by antioxidant defense systems. Cells produce ROS as a control of physiological processes, but increasing ROS becomes pathological leading to non-specific and irreversible damage to biological molecules, such as DNA, lipid, and protein. Endogenous ROS are mainly produced by mitochondria during both physiological and pathological conditions, and enzymes, such as nicotinamide adenine dinucleotide phosphate oxidase (NOX), xanthine oxidase (XO), lipoxygenase (LOX), myeloperoxidase (MPO) and monoamine oxidase (MAO). To neutralize ROS, the body employs enzymatic and non-enzymatic defense systems. The dietary intake of bioactive phenols, such as quercetin (Que), is a non-enzymatic system that can quench ROS and protect from pro-oxidative damage. In this review, we evaluate the ability of Que to target endogenous oxidant enzymes involved in ROS production and explore the mechanisms of action underlying its antioxidant properties. Que not only acts as a free radical scavenger by donating electrons through the negative charges in its phenolic and ketone groups, but it can effectively inhibit the activity of several endogenous oxidative enzymes, binding them with high affinity and specificity. Among all targets, Que showed the best results in molecular docking simulations with XO, followed by MAO-A, 5-LOX, NOX, and MPO. Taken together, these findings highlight the potential of Que as a natural antioxidant therapy for oxidative stress-related diseases.

Solid state fermentation (SSF) is considered more sustainable than traditional fermentation because it uses low amounts of water and transforms agro-industrial residues into value added products. Enzymes, biofuels, nanoparticles and bioactive compounds can be obtained from SSF. The key factor in SSF processes is the choice of microorganisms and their substrates. Many fungal species can be used and are mainly used due their lower requirements of water, O2 and light. Residues rich in soluble and insoluble fiber are utilized by lignocellulolytic fungi because they have the enzymes that break fiber hard structure (lignases, celullases or hemicelullases). During the hydrolysis of lignin, some phenolic compounds are released but fungi also synthetize compounds such as mycophenolic acid, dicerandrol C, phenylacetates, anthraquinones, benzofurans and alkenyl phenols that have health beneficial effects such as antitumoral, antimicrobial, antioxidant and antiviral activities. Another important group of compounds synthetized by fungi during fermentation are polysaccharides that also have important health promoting properties. Fungal biofermentation has also proved to be a process which can release high contents of phenolics and it also increases the bioactivity of these compounds.

No-till and cereal-legume intercropping have been recognized as favorable cropping practices to increase crop yields while maintaining soil quality in arid and semiarid environment, but the bio-logical mechanisms are poorly understood. The present study was to determine the response of soil properties, enzyme activities, and microbial community diversity and composition in mono- and inter-cropping under conventional and no-tillage conditions. We initiated a field experiment in Wuwei, a typical arid area of China, in 2014. Soil was sampled in August 2022 and, yields, soil properties, enzyme activities, and the microbial community diversity and composition were de-termined in the maize and pea strips in inter- and mono-cropping systems. Results revealed that the maize and pea strips in the no-till intercropping significantly increased yields, total and or-ganic carbon stocks, decreased NO3--N, and obtained the highest total and organic P in the soil. The α- and β-diversity of archaea and eukaryotes were significantly affected by planting patterns, while α- and β-diversity of the bacterial community were significantly affected by tillage practices. Both no-tillage and intercropped maize significantly increased the abundance of archaea phylum Thaumarchaeota and bacterial phylum Nitrospirae, benefiting nitrogen fixation of intercropped pea from the atmosphere under the no-tillage cereal/legume intercropping. No-till intercropping was conducive to the accumulation of organic carbon, while decreasing the abundance of Prote-obacteria, Acidobacteria, and Verrucomicrobia. Limited soil enzyme activities (ACP, ALP, DP, NAG, BG, AG, CB) led to decreases in organic carbon turnover and utilization. Intercropping al-tered soil microbial community diversity and composition due to changes in soil properties and enzyme activities. These findings suggest that no-tilled cereal-legume intercropping is a sustaina-ble cropping practice for improving soil properties and enhancing microbial (archaea, bacterial, Eukaryota) diversity, but the long-term persistence is not conducive to rapid turnover of soil nu-trients due to limited enzyme activities.

Recently, two respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We developed and validated an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and choice of blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 μg/mL of DS-Cav1 and SC-TM, respectively, showed high coefficients of determination (r2 = 0.90). Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r2 = 0.90) and SC-TM (r2 = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM but not with the attachment protein.

NADPH is an indispensable cofactor in a wide range of physiological processes which is generated by a family of NADPH-dehydrogenases, where the NADP-dependent malic enzyme (NADP-ME) is one member of these enzymes. Pepper (Capsicum annuum L.) fruit is a horticultural product worldwide consumed which has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteome ic, biochemical, and metabolic levels. Nitric oxide (NO) is a recognized signal molecule that can exert regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Based on a data-mining approach on the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time-course expression analysis of these genes during different fruit ripening stages including green immature (G), breaking point (BP), and red ripe (R) showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. Furthermore, the analysis of the cis-regulatory elements showed that CaNADP-ME2 and CaNADP-ME5 were positively regulated by the light-responsive element Box4 whereas CaNADP-ME4 was by ABRE (ACGT-containing abscisic acid response element), which is involved in the abscisic acid (ABA) responsiveness. To get deeper knowledge on this enzyme system from fruits, we obtained the 50-75% ammonium-sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allowed identifying four isozymes designated CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and by exogenous NO gas treatment.

Enzyme replacement therapy (ERT) improves the somatic manifestations in mucopolysaccharidoses (MPS))．However, because intravenously administered enzymes cannot cross the blood brain barrier (BBB), ERT is ineffective against the progressive neurodegeneration and resultant severe central nervous system (CNS) symptoms observed in patients with neuronopathic MPS. Attempts to surmount this problem have been made with intrathecal and intracerebroventricular ERT intend to achieve CNS effects, but the burdens on patients are inimical to long-term multiple administrations. However, after pabinafusp alfa, a human iduronate-2-sulfatase fused with a BBB-crossing anti-transferrin receptor antibody, showed both central and peripheral efficacy in mice model, subsequent clinical trials in a total of 62 patients with MPS-II (Hunter syndrome) in Japan and Brazil substantiated this dual efficacy and provided an acceptable safety profile. To date, pabinafusp alfa is the only approved intravenous ERT effective against both the somatic and CNS symptoms of patients with MPS-II. This article summarizes the hitherto obtained preclinical and clinical evidence associated with this drug, and discusses the preclinical, translational and clinical challenges of evaluating, ameliorating and preventing the neurodegeneration in patients with MPS-II.

The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC50 value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behaviour remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.

The angiotensin-converting enzyme (ACE) is a peptidase that is involved in the synthesis of Angiotensin II, the bioactive component of the renin-angiotensin system. A growing body of literature argues for a beneficial impact of ACE inhibitors (ACEi) on age-associated metabolic disorders, mediated by cellular changes in reactive oxygen species (ROS) that improve mitochondrial function. Yet, our understanding of the relationship between ACEi therapy and metabolic parameters is limited. Here, we used three genetically diverse strains of Drosophila melanogaster to show that Lisinopril treatment reduces thoracic ROS levels and mitochondrial respiration in young flies, and increases mitochondrial content in middle-aged flies. Using untargeted metabolomics analysis, we also showed that Lisinopril perturbs the thoracic metabolic network structure by affecting metabolic pathways involved in glycogen degradation, glycolysis, and mevalonate metabolism. The Lisinopril-induced effects on mitochondrial and metabolic parameters, however, are genotype-specific and likely reflect the drug’s impact on nutrient-dependent fitness traits. Accordingly, we found that Lisinopril negatively affects survival under nutrient starvation, an effect that can be blunted by genotype and age in a manner that partially mirrors the drug-induced changes in mitochondrial respiration. In conclusion, our results provide novel and important insights into the role of ACEi in cellular metabolism.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here we reported that the FDA-approved, oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammation stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of antioxidant reduced glutathione and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of critical histone methyltransferase, Enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant genes regulatory regions, in an antioxidative response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression and modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD and represents a therapeutic strategy for further development.

Salmonella causes various type of disease worldwide with a remarkable pace. Salmonella enteric serotype typhi (S. typhi), a gram-negative bacterium (only cause disease in man) is the mainly causative agent of Typhoid fever. Typhoid fever is most common in poor and unprivileged developing countries of Asia and Africa. One of the major components of virulence factors produced during salmonella infection is Lipid A, which acts as a potent human immuno-modulator bacterial endotoxin. Regulation of Lipid A biosynthetic pathway occurs at second step, catalyzed by LpxC, a Zn2+ dependent metalloamidase. Systematic Screening of a pool of drug datasets like natural products library from Zinc database, Asinex database, Thiophene analogues fruitfully provided us 3 potent lead molecules s1_dl_mseq2, s1_ll_mseq2, and s2_ll_mseq8 which actively binds with LpxC enzyme and could be developed into sound inhibitors of LpxC enzyme after the application of drug development and processing strategies. Wet lab experimentation is required to validate these results for further use.

Protein–nanoparticle hybridization can ideally lead to novel biological entities characterized by emerging properties, which can sensibly differ from those of the parent components. Herein, the effect of ionic strength on the biological functions of recombinant, His-tagged spermine oxidase (i.e., SMOX) was studied for the first time. Moreover, SMOX was integrated to colloidal surface active maghemite nanoparticles (SAMNs) by direct self-assembly, leading to a biologically active nano-enzyme (i.e., SAMN@SMOX). The hybrid was subjected to an in-depth chemical-physical characterization highlighting that protein structure was perfectly preserved. The catalytic activity of the nanostructured hybrid (SAMN@SMOX) was assessed by extracting kinetics parameters using spermine as substrate and compared to the soluble enzyme as a function of ionic strength. Results revealed that the catalytic function was dominated by electrostatic interactions and that they were drastically modified upon hybridization with colloidal ɣ-Fe2O3. Noteworthy, the affinity of SMOX toward spermine was significantly higher for the nanohybrid at low salinity. The present study supports the vision of using protein–nanoparticle conjugation as a mean for modulating biological functions.

CD44 is a type I transmembrane glycoprotein, and possesses various isoforms which are largely classified into CD44 standard and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 mAb, C₄₄Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C₄₄Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10), and found that C₄₄Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C₄₄Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C₄₄Mab-108 includes Asp280 and Trp281 of CD44v3-10.

Genome-scale metabolic models (GEMs) play an important role in the phenotype prediction of microorganisms, and their accuracy can be further improved by integrating other types of biological data such as enzyme concentrations and kinetic coefficients. Enzyme-constrained models (ecModels) have been constructed for several species and were successfully applied to increase the production of commodity chemicals. However, there was still no genome-scale ecModel for the important model organism Bacillus subtilis prior to this study. Here, we integrated enzyme kinetic and proteomic data to construct the first genome-scale ecModel of B. subtilis (ecBSU1) using the ECMpy workflow. We first used ecBSU1 to simulate overflow metabolism and explore the trade-off between biomass yield and enzyme usage efficiency. Then, we simulated the growth rate on eight previously published substrates and found that the simulation results of ecBSU1 were in good agreement with the literature. Finally, we identified target genes that enhance the yield of commodity chemicals using ecBSU1, most of which were consistent with the experimental data, and some of which may be potential novel targets for metabolic engineering. This work demonstrates that the integration of enzymatic constraints is an effective method to improve the performance of GEMs. The ecModel can predict overflow metabolism more precisely and can be used for the identification of target genes to guide the rational design of microbial cell factories.

The construction of optimized biological fuel cells requires a cathode which combines the longevity of a microbial catalyst with the power density of an enzymatic catalyst. Laccase secreting fungi were grown directly on the cathode of a biological fuel cell to facilitate the exchange of inactive enzymes with active enzymes with the goal of extending the lifetime of laccase cathodes. Additionally, a functionally graded coating was developed to increase enzyme loading at the cathode. Directly incorporating the laccase producing fungus at the cathode extends the operational lifetime of laccase cathodes while eliminating the need for frequent replenishment of the electrolyte. Additionally, the hybrid microbial-enzymatic cathode addresses the issue of enzyme inactivation by using the natural ability of fungi to exchange inactive laccases at the cathode with active laccases. Finally, enzyme adsorption was increased through the use of a functionally graded coating containing an optimized ratio of titanium dioxide nanoparticles and single walled carbon nanotubes. The hybrid microbial-enzymatic fuel cell combines the higher power density of enzymatic fuel cells with the longevity of microbial fuel cells and demonstrates the feasibility of a self-regenerating fuel cell in which inactive laccases are continuously exchanged with active laccases.

Emergence of multi drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis poses a serious threat to TB control as the available treatment options are less effective in these cases. Therefore, new strategies are required for identification of new drugs and drug targets. The M. tuberculosis dihydrodipicolinate synthase is a putative drug target with no potent inhibitor. Here in this study, we have used a comprehensive computational approach to identify small molecule inhibitors and validated them in vitro. As similar structures tend to have similar functions, therefore 3 approaches, namely, (a) known inhibitor-based approach, (b) substrate analogues, (c) product analogues were used for compound screening from various chemical libraries. The fourth approach we have taken is fragment library screening.In vitro studies revealed that β-hydroxypyruvic acid tartronic acid are inhibitors of Mtb-rDapA with maximum inhibition 48% and 51% at 500µM concentrations respectively. We have also observed that β-hydroxypyruvic acid is a time dependent inhibitor of Mtb-rDapA. Further, thermal shift assays showed that β-hydroxypyruvic acid interacts with Mtb-rDapA and the thermal stability of Mtb-rDapA shifted by 3˚C. Among the product analogues of Mtb-DapA, dipicolinic acid showed to bind to Mtb-rDapB shifting thermal stability of Mtb-rDapB by 0.5˚C in binding. Additionally, we have designed a new approach by combining substrate and product analogues to achieve better inhibitions. In vitro assays validated that this combination approach increased maximum inhibition up to 100%. We envision that these compounds would serve as potent leads for tuberculosis drug discovery.

Breast cancer (BC) is the most common malignancy and the second cause of cancer specific death in women from high-income countries. More than 70% of all breast cancer are hormone receptor-positive BC, and elevated estrogen circulating in blood has been proved to be a strong risk for BC development. This is due to its contribution to enhance proliferation of cancerous cells, angiogenesis and metastasis stimulation and therapy resistance. The estrogen (E) metabolism–gut microbiome axis is functional with underlying individual variations in E levels. It is reasonable that the estrobolome (bacterial genes whose products are capable of metabolizing E) could contribute to the risk of hormone-driven malignancies including BC and may serve as a potential biomarker and target. Gut microbial β -glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome. Furthermore, bacterial GUS enzymes within the gastrointestinal tract have been postulated to contribute to hormone breast cancer. In this review, we discuss the recent knowledge about the role of GUS enzyme in the pathogenesis of breast cancer. We focus on the role of GUS in (i)-the microbioma and estrogen metabolism, (ii) diet, estrobolome, and the BC development, (iii) other activities of the bacterial β-glucuronidase, and (iv) the new molecular target for BC therapeutic application.

Rice bran arabinoxylan compound (RBAC) is derived from rice bran enzymatically treated with Lentinus Edodes mycelium. This review explores biologically active compounds and mechanisms of action that support RBAC as an immunomodulating nutraceutical in generally healthy and/or aging individuals. Thirty-seven (n=37) primary research articles fulfiled the selection criteria for review. Most research is based on Biobran MGN-3, which consists of complex heteropolysaccharides with arabinoxylan as its primary structure while also containing galactan and glucan. RBAC was found to invoke immunological activities through direct absorption via the digestive tract and interaction with immune cells at the Peyer’s patches. RBAC was shown to promote the innate defence by upregulating the macrophage phagocytosis and enhancing natural killer cell activity while lowering oxidative stress. Through induction of dendritic cell maturation, RBAC also augments adaptive immunity by promoting T and B lymphocyte proliferation. RBAC acts as an immunomodulator by inhibiting mast cell degranulation during allergic reactions, attenuating inflammation, and downregulating angiogenesis by modulating cytokines and growth factors. RBAC is shown to be a safe and effective neutraceutical for improving immune health, notably in aging individuals with reduced immune function. Human clinical trials with geriatric participants have demonstrated RBAC to have prophylactic benefits against viral infection and may improve their quality of life. Further research should explore RBAC’s bioavailability, pharmacodynamics, and pharmacokinetics of the complex heteropolysaccharides within. Translational research to assess RBAC as an nuetraceutical for the aging population is still required, particularly in human studies with larger sample sizes and cohort studies with long follow-up periods.

Pretilachlor is a chloroacetamide herbicide, mainly used for the control of weeds and broadleaf weeds in rice, and widely used in China. For the detection of residues of pretilachlor in the en-vironment and food, a highly sensitive and specific monoclonal antibody against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47±2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed for the detection of pretilachlor residues in the environment and food crops. The specificity of the antibody was explained by computer simula-tions and experimental validation. No cross-reactivity of this monoclonal antibody to alachlor, acetochlor and metalaxyl, and it cross-reacted less than 3.0% to both butachlor and propisochlor. The limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3%~91.3%. The ic-ELISA method was validated by high-performance liquid chromatography, thus, it can be used for residue detection of pretilachlor in the environment and grains.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson´s and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding on this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass-spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer and with FAD and dicoumarol ligand binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, valuable to understand at the molecular level the effects of disease-associated variants, post-translation modifications and ligand binding cooperativity in NQO1.

Nuts are a staple food consumed throughout the world, and their purity is essential to maintaining human health. However, fungal mycotoxins pose one of the real challenges in keeping nuts clean for human consumption. The high cost of equipment, reagents used, standard samples, as well as increased requirements for personnel qualifications limit the use of chromatographic methods in testing laboratories, both manufacturing enterprises and organizations monitoring food safety. The present study aimed to test a new method for extracting aflatoxin B1 using sodium chloride and citric acid instead of methanol present in imported nuts obtained from local markets in Astana (Kazakhstan). The proposed method for extracting aflatoxin B1 from nuts is chemically safe, since the substances used belong to hazard class 3 of the international classification and do not require separate premises in food safety laboratories of the Astana markets and special training of specialists. The technical objective of the method is to replace the reagent (methanol) during the sample preparation process when conducting cocuret enzyme-linked immunosorbent assay. Aflatoxin B1 levels varied depending on the type of nut. Aflatoxin contamination was observed in 35.8% of samples analyzed. These levels of mycotoxin contamination are alarming and raise serious questions about the purity of imported nuts to ensure food safety.

Glycerol has found its applications as a heat-transfer fluid in heat exchangers, and as a compo-nent of functional solutions in biosensor analysis. Flowing non-aqueous fluids are known to be able to induce electromagnetic fields due to triboelectric effect. These triboelectrically generated electromagnetic fields can affect biological macromolecules. Horseradish peroxidase (HRP) is widely employed as a convenient model object for studying how external electric, magnetic, and electromagnetic fields affect enzymes. Herein, we have studied whether the flow of glycerol in a ground-shielded cylindrical coil affects the HRP enzyme incubated at a 2 cm distance near the coil’s side. Atomic force microscopy (AFM) has been employed in order to study the effect of glycerol flow on HRP at the nanoscale. Increased aggregation of HRP on mica has been observed after the incubation of the enzyme near the coil. Moreover, the enzymatic activity of HRP has also been affected. The results reported can find their application in biotechnology, food technology and life sciences application, considering the development of triboelectric generators, en-zyme-based biosensors and bioreactors with surface-immobilized enzymes. Our data can also be of interest for scientists studying triboelectric phenomena.

Fabry disease is a lysosomal storage disorder caused by the deficiency of the α-galactosidase-A enzyme. Cardiac, renal, and neurological involvement significantly reduces life expectancy. Until a few years ago, treatment options for Fabry disease were limited to enzyme replacement therapy with agalsidase alfa or beta administered by intravenous infusion every 2 weeks. Migalastat (Galafold®) is an oral pharmacological chaperone that increases enzyme activity of “amenable” mutations. The safety and efficacy of migalastat were supported in the phase III FACETS and ATTRACT studies, compared to available enzyme replacement therapies; showing a reduction in left ventricular mass, and stabilization of kidney function and plasma Lyso-Gb3. Similar results were confirmed in subsequent extension publications, both in patients who started migalastat as their first treatment and in patients who were previously on enzyme replacement therapy and switched to migalastat. In this review we describe the safety and efficacy of switching from enzyme replacement therapy to migalastat in patients with Fabry disease and “amenable” mutations, referring to publications available to date.

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia epidemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2, predicting potential intermediate hosts of SARS-CoV-2.

Development of carbon nanomaterials for adsorption thus removal of organic pollutants from water is a progressive research subject. In this regard, carbon nanomaterials with bifunctionality towards polar and non-polar or even amphiphilic undesired materials is indeed attractive for further study and implementation. Here, we created carbon buckypaper adsorbents comprising amphiphilic (oxygenated amorphous carbon (a-COx)/graphite (G)) nanofilaments that can dynamically adsorb organic biomolecules (i.e. urease enzyme) and thus purify the wastewaters of relevant industries. Given the dynamic conditions of the test, the adsorbent was highly efficient in adsorption of the enzyme (88%) while permeable to water (2382 L.h-1.m-2), thus holds a great promise for further development and upscaling. A subsequent citric acid functionalization declined selectivity of the membrane to urease, implying the biomolecules adsorb mostly via graphitic domains rather than oxidized, polar amorphous carbon ones. The devised platform i.e. the urease functionalized buckypaper is optimally conductive (13 S.cm-1) and can be further employed as a biosensor. Accordingly, water treatment can be linked to biosensing via a nanostructured membrane.

a Special Issue of Microorganisms devoted to ‘Microbial Biocatalysis and Biodegradation’ would be incomplete without some form of acknowledgement of the many important roles that dioxygen-dependent enzymes (principally mono- and dioxygenases) play in relevant aspects of biooxygenation. This is reflected by the multiple strategic roles that dioxygen -dependent microbial enzymes play both in generating valuable synthons for chemoenzymatic synthesis, and in facilitating key reactions that drive the global geochemical Carbon Cycle. A useful insight into this relationship can be gained by reviewing the evolution of the current status of 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) from camphor-grown Pseudomonas putida ATCC 17453. Over the last sixty years, the perceived nature of this monooxygenase has transmogrified significantly. Commencing in the 1960s, extensive initial studies consistently reported that the enzyme was a monomeric true flavoprotein dependent on both FMNH₂ and nonheme iron as bound cofactors. However, over the last decade all those criteria have changed absolutely, and the enzyme is currently acknowledged to be a metal ion-independent homodimeric flavin-dependent two-component monooxygenase deploying FMNH₂ as a cosubstrate. That transition is a paradigm of the ever evolving nature of scientific knowledge.

Background: This study analyzes sex-based differences in renal structure and response to the Angiotensin-Converting Enzyme (ACE) inhibitor enalapril in a mouse model of atherosclerosis. Methods: ApoE-/- mice (8 weeks old) received enalapril (5 mg/kg/day, subcutaneous) or PBS as a control for an additional 14 weeks. Each group consisted of six males and six females. Results: Females exhibited elevated LDL-cholesterol levels, while males presented higher creatinine levels and proteinuria. Enalapril effectively reduced blood pressure in both groups, but proteinuria decreased significantly only in females. Plaque size analysis and assessment of kidney inflammation revealed no significant sex-based differences. However, males displayed more severe glomerular injury, with increased mesangial expansion, mesangiolysis, glomerular foam cells and activated parietal epithelial cells (PECs). Enalapril mitigated mesangial expansion, glomerular inflammation (particularly in females), and the hypertrophy of PECs in males. Conclusion: This study demonstrates sex-based differences in the response to enalapril in a mouse model of atherosclerosis. Males exhibited more severe glomerular injury, while enalapril provided renal protection, particularly in females. These findings suggest potential sex-specific considerations for ACE inhibitor therapy in chronic kidney disease and atherosclerosis cardiovascular disease. Further research is needed to elucidate the underlying mechanism behind these observations.

In traditional oriental medicine, carrots (Daucus carota L.) are considered effective medicinal herbs; however, the use of D. carota leaves (DGL) as therapeutic agents has not been explored in depth. Therefore, we aimed to demonstrate the value of DGL, generally treated as waste while developing plants for wide industrial availability. Six flavone glycosides were isolated and identified from DGL, and their constituents were identified and quantitated using an NMR and HPLC/UV method, which was optimized and validated. The structure of chrysoeriol-7-rutinoside from DGL was elucidated for the first time. The method exhibited adequate relative standard deviation (<1.89%) and recovery (94.89–105.97%). The deglycosylation of DGL flavone glycosides by Viscozyme L and Pectinex was assessed. Upon converting the reaction contents to percentages, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively. The enzyme-treated DGL had a higher inhibitory effect on TNF-α and IL-2 expression than that of the carrot roots or carrot leaves without enzyme treatments. These results highlight the importance of carrot leaves and could be used as baseline standardization data for commercial development.

The purpose of this study was to develop an in-vitro digestion protocol to evaluate the antioxidant potential of the peptides found in processed cheddar cheese using digestion enzymes. We studied first antioxidant and angiotensin converting enzyme (ACE) inhibition and antioxidant activities of processed cheddar cheese with the addition of spices e.g. cumin, clove and black pepper made from buffalo milk and ripened for 9 months. Then we conducted an in vitro digestion of processed cheddar cheese by gastric and duodenal enzymes. Freeze dried water (WSE) and ethanol soluble fractions (ESE) of processed cheddar cheese were also monitored for their ACE inhibition activity and antioxidant activities. In our preliminary experiments, different levels of spices (cumin, clove and black pepper) were tested into cheese matrix and only one level 0.2g/100g (0.2%) on the basis of cheese weight was considered good concerning sensory evaluation. Significant increase in ACE-inhibition (%) of processed Cheddar cheese as well as its WSE and ESE was obtained. Lower IC50 values were found after duodenal phase digestion compared to oral phase digestion.

Galanthus elwesii Hook is an important plant species of the Amaryllidaceae family and is used for medicinal purposes with its valuable bioactive compounds. The present study was conducted to investigate the effects of foliar salicylic acid (SA) and zinc (Zn) treatments on proline, carotenoid, chlorophyll content and anti-oxidant enzyme activity in G. elwesii. The ascorbate peroxidase (APX), catalase (CAT) enzyme activity, and protein contents were determined with ascorbate oxidation, hydrogen peroxide (H2O2), and Bradford experiments, respectively. The plants were treated with three different concentrations of SA (0.5, 1, and 2 mM) and, and Zn (40, 80, and 120 mM) were compared with the control. Fresh leaves were harvested in the study. APX (3.99 ± 0.58 EU/mg protein) and CAT (154.64 ± 4.10 EU/mg protein) were obtained from Zn 80 and 120 mM treatments at the highest level, respectively. Proline, chlorophyll b, and carotenoid content increased 12.4, 1.54 and 3.95-fold, respectively, in 0.5 mM SA treatments when matched with control group. It was found that increasing doses of SA and Zn increased the content of malondialdehyde (MDA), but this was not at a significant level. The total chlorophyll content increased 2.27-fold in Zn 120 mM + SA 2 mM treatment and the chlorophyll content increased 2.41-fold in Zn 40 mM + SA 1 mM treatment.

The adsorption of proteins on polymer is widely used in biosensors. Here, molecular dynamics (MD) simulation was used to study the immobilization of angiotensin converting enzyme II (ACE2) with six initial orientations proposed on polystyrene (PS) at the ambient conditions of pH (4.5, 6, 7, 8, 9.5) and NaCl (0.01, 0.05, 0.1, 0.15, 0.2, 0.25 M). ACE2 immobilization under favorable ambient conditions was characterized by minimum distance (short), settlement time (fast), interaction energy (substantial) and protein configuration (stable). ACE2 orientations proposed in 0.15M NaCl were respectively preferable to (90, 0, 0), (0, 0, 0) and (0, 270, 0), (180, 0, 0) and (0, 90, 0), (90, 0, 0) at pH 4.5, 6, 7, 9.5. ACE2 immobilization was further evaluated at pH 7 by optimizing NaCl concentration. Its proposed orientations of (i) (0, 270, 0), (ii) (0, 0, 0) and (90, 0, 0), and (iii) (0, 90, 0) and (90, 0, 0) were preferable in 0.05, 0.1 and 0.2 M NaCl, respectively. The great significance of cubic-orientation settlement mode provides tangible improvement for the microfabrication of biochips for rapid diagnosis of severe acute respiratory syndrome coronavirus II (SARS-CoV-2).

Epithelial cells can undergo apoptosis by manipulating the balance between pro-survival and apoptotic signals. In this work, we show that TRAIL-induced apoptosis can be differentially regulated by the expression of α(1,6)fucosyltransferase (FucT-8), the only enzyme in mammals that transfers the (1,6)fucose residue to the pentasaccharide core of complex N-glycans. Specifically, in the cellular model of colorectal cancer (CRC) progression formed using the human syngeneic lines SW480 and SW620, knockdown of the FucT-8-encoding FUT8 gene significantly enhanced TRAIL-induced apoptosis in SW480 cells. However, FUT8 repression did not affect SW620 cells, which suggests that core fucosylation differentiates TRAIL-sensitive premetastatic SW480 cells from TRAIL-resistant metastatic SW620 cells. In this regard, we provide evidence that phosphorylation of ERK1/2 kinases can dynamically regulate TRAIL-dependent apoptosis and that core fucosylation can control the ERK/MAPK pro-survival pathway in which SW480 and SW620 cells participate. Moreover, the depletion of core fucosylation sensitises primary tumour SW480 cells to the combination of TRAIL and low doses of 5-FU, oxaliplatin, irinotecan or mitomycin C. In contrast, combination of TRAIL and oxaliplatin, irinotecan or bevacizumab reinforces resistance of FUT8-knockdown metastatic SW620 cells to apoptosis. Consequently, FucT-8 could be a plausible target for increasing apoptosis and drug response in early CRC.

Salmonella enterica includes enteric pathogens of zoonotic potential, possessing one of the largest pools of temperate phages in their genomes. One such class of phages previously called Podoviridae (Now genera placed directly under class Caudoviricetes temporarily) is the largest group of temperate phages that lysogenize clinically and economically important Salmonella enterica serovars. These phages are capable of generalized transduction and are well known for carrying foreign DNA (mobile genetic elements and resistance genes) to new bacterial species that play a major role in the evolution, pathogenicity, and host adaptability of Salmonella serovars. Here we report a novel species of podo viruses; Salmonella phage BIS08P22 (BIS08) that infects Salmonella enterica serovar Typhimurium strain SE-BS17 (Acc. NO: MZ503545). Phage BIS08 was viable only at biological pH and temperature (pH7 and 37 °C) and features 41,574 -base pair (bp) linear ds DNA genome with 47% GC content. It encodes 73 putative Open Reading Frames (ORFs), has a mosaic arrangement, and shares only 16 core genes with its closest homologs. BIS08 genome possesses only 52 % homology (genome-wide Nucleotide homology by VIRIDIC analysis) with members of the genus Lederbergvirus which is not sufficient to place it in the same genus. This phage has a unique terminase enzyme (DNA packaging motor) with no nucleotide and protein homology with any known member of the Lederbergvirus genus. We carried out a detailed phylogenetic and genome analysis of this novel phage and proposed its placement in a new genus. Our study also suggests a revision in the classification of the genus Lederbergvirus. We also performed in silico structural analysis of the BIS08 unique terminase enzyme comparing it to its close homologs.

COS production is carried out enzymatically using chitosanase, but this process is expensive. Production costs can be reduced by using crude enzyme chitosanase, and the resulting COS has depolymerization < 20 and molecular weight < 3900 Da. Sources of crude enzyme chitosanase come from the soil, sediment, and fishery waste. Chitosanase is produced from bacteria and molds isolated from the source or direct isolation process for extracellular enzymes, especially from fishery waste. Microorganisms in the soil produce chitosanase as a biocontrol and self-defense agent. Bacteria and molds have been isolated, including Bacillus cereus, Bacillus mycoides, Paenibacillus sp., and Penicillium janthinellum. Fishery waste, especially parts of the digestive tract, is a source of chitosanase because of its relation to its nature and food, such as small fish, frogs, and several types of crustaceans. In addition, fishery waste can be used as a carbon and nitrogen source (C/N) in microorganism culture media. The chitin content becomes a stimulant for the synthesis of chitosanase by microorganisms. Crude enzyme chitosanase has an active site that hydrolyzes chitosan by breaking glycosidic bonds and producing glucosamine units (GlcN) and N-acetyl glucosamine (GlcNAc). This mechanism can take place through two events, namely inverting and retaining. Crude enzyme chitosanase can break glycosidic bonds, especially with the same groups, for example, bonds between glucosamine or N-acetyl glucosamine. The COS produced is of the partially acetylated COS (paCOS) type, which still contains an acetyl group. The degree of deacetylation is a significant factor in the resulting COS product, COS with the fully deacetylated COS (fdCOS) type of chitosan with a high degree of deacetylation. Other factors to consider are the raw material of chitosan, the incubation time, the screening process, and the production of microorganism cultures.

The study addresses the management of kitchen waste by transforming it into biofertilizer formulations utilizing an effective in-house developed multi-enzyme preparation. A separate hydrolysis and fermentation bioprocessing approach which in particular relates to the application of a separate enzyme preparation from Aspergillus niger P-19 to separately hydrolyze kitchen waste, followed by the growth of Klebsiella pneumoniae AP-407 in the liquid hydrolysate as well with simultaneous production of carrier-based biofertilizer. In liquid and carrier-based biofertilizers, the viable cell count reaches 3.00 × 1012 CFU/ml and 3.00 × 1012 CFU/g, respectively. The current study adopts a novel process technique for the manufacturing of both carrier and liquid biofertilizers, adopting a zero-waste approach to the management of kitchen waste.

The present study was conducted to evaluate the effects of dietary supplementation of tea polyphenol （TP） on serum hormone, serum enzyme activity, antioxidant-related and immune-related gene expression of laying hens under heat stress. A total of 288 Chinese yellow chicken (186 days old) were randomly distributed among two treatments, each of which included 6 replicates of 24 hens. Dietary treatments were that the basal diet was supplemented with 200 mg / kg tea polyphenol. The study lasted for 7 weeks, including 1 week of adaptation and 6 weeks of the formal test. The content of high-density lipoprotein cholesterol (HDL-C) and total protein (TP) in serum significantly decreased by dietary supplementation with tea polyphenol. Dietary tea polyphenol supplementation improved serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) enzyme activity and decreased serum malondialdehyde (MDA) content in treatments compared to the control. However, supplementation of tea polyphenol did not affect the activity of serum catalase (CAT). The results indicated that long-term feeding of tea polyphenols help to increase the amount of hormones (FSH, E2) associated with reproduction in laying hens and thus improve egg production. It also improved the immune function of laying hens in high temperature environments. Adding tea polyphenols to the diet can significantly increase the serum IgG, IgM content of the laying hens and can upregulate the IgA content. Dietary supplementation of tea polyphenols in the laying hens significantly increased the expression of antioxidant enzyme-related genes (SOD, CAT and GPX1) in the liver. Moreover, the addition of tea polyphenols significantly increased the expression of immune-related genes (Interferon-γ (INF-γ), Interleukin 2 (IL-2) and Interleukin 4 (IL-4)) in the spleen. It is concluded that addition of tea polyphenols has a positive effect on antioxidant activity and immune function of laying hens.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict a SAM binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization by assays diagnostic for loss of diphthamide reveal the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5′-deoxyadenosyl (dAdo) radicals.

Aims: Angiotensin-converting enzyme 2 (ACE2) plays an important role in the entry of coronaviruses into host cells. This paper described how carnosine, a naturally occurring supplement, can be an effective drug candidate for coronavirus disease (COVID-19) on the basis of molecular docking and modeling to host ACE2 co-crystallized with COVID-19 spike protein. Methods: First, the starting point was ACE2 inhibitors and their structure-activity relationship (SAR). Next, chemical similarity (or diversity) and PubMed searches made it possible to repurpose and assess approved or experimental drugs for COVID-19. In parallel, at all stages, authors performed bioactivity scoring to assess potential repurposed inhibitors at ACE2. Finally, investigators performed molecular docking and modeling of the identified drug candidate to host ACE2 co-crystallized with COVID-19 spike protein. Results: Carnosine emerged as the best known drug candidate to match ACE2 inhibitor structure. Preliminary docking was more optimal to ACE2 than the known typical angiotensin-converting enzyme 1 (ACE1) inhibitor (enalapril) and quite comparable to known or presumed ACE2 inhibitors. Viral spike protein elements binding to ACE2 were retained in the best carnosine pose in SwissDock at 1.75 Angstroms. Out of the three main areas of attachment expected to the co-crystallized protein structure, carnosine bind with higher affinity to two compared to the known ACE2 active site. LibDock score was 92.40 for site 3, 90.88 for site 1, and inside the active site 85.49. Conclusion: Carnosine has promising inhibitory interactions with host ACE2 co-crystallized with COVID-19 spike protein and hence could offer potential mitigating effect against current COVID-19 pandemic.

Staphylococcal bacteriophages of Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs, IsaA and SceD. We show that Tgl is also a lytic enzyme secreted by bacterial transport system and localizes to cell peripheries, like IsaA and SceD. It caused lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destruct S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with the decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from gene lysK, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, as are phage early genes. Taken together our data indicate that tgl is a part of kayviruses lytic module and encodes an additional endolysin which can act in concert with LysK in cell lysis.

Background. The characterization of new biomarkers of COVID-19 is extremely important. Few studies measured the soluble receptor for advanced glycation end product (sRAGE), angiotensin-converting enzyme 2 (ACE2), calcium and magnesium in COVID-19. Aims: To measure sRAGE, ACE2, interleukin (IL)-6, IL-10, CRP, calcium, magnesium, and albumin in COVID-19 patients in association with peripheral oxygen saturation (SpO2) and chest CT scan abnormalities (CCTA) including ground glass opacities. Methods. This study measured sRAGE, ACE2, IL-6, IL-10, CRP using ELISA techniques, and calcium, magnesium, and albumin using a spectrophotometric method in 60 COVID-19 patients and 30 healthy controls.Results. COVID-19 is characterized by significantly increased IL-6, CRP, IL-10, sRAGE, ACE2, and lowered levels of SpO2, albumin, magnesium and calcium. Neural networks showed that a combination of calcium, IL-6, CRP, and sRAGE yielded an accuracy of 100% in detecting COVID-19 patients with calcium being the most important predictor followed by IL-6, and CRP. COVID-19 patients with CCTAs showed lower SpO₂ and albumin levels than those without CCTAs. SpO2 was significantly and inversely correlated with IL-6, IL-10, CRP, sRAGE, and ACE2, and positively with albumin, magnesium and calcium. Patients with positive IgG results showed a significant elevation in the serum level of IL-6, sRAGE, and ACE2 compared to the negatively IgG patient subgroup.Conclusion. The results show that immune-inflammatory and RAGE pathway biomarkers may be used as external validating criterion for the diagnosis COVID-19. Those pathways coupled with lowered SpO2, calcium and magnesium are drug targets that may help to reduce the consequences of COVID-19.

Cyanobacterial mass developments in eutrophic ponds and lakes are a major concern for lake management, as many cyanobacteria produce a huge variety of toxic secondary metabolites, e.g. microcystins. The aim of this research was to culture a strain of the cyanobacterium Microcystis sp strain BM25, to observe its biomass production and to isolate and purify protease inhibitors from this cyanobacterial biomass. Different secondary metabolites were isolated following a standard bioassay-guideline. Isolation was performed, with an enzymatic protease assay as bioassay. High performance liquid chromatography was used to identify different fractions of secondary metabolite from the strain BM25. Moreover, protease homogenates were isolated from Daphnia magna in order to test the inhibitors against naturally occurring major digestive proteases trypsin and chymotrypsin. It was measured that 60% MeOH and the 80% MeOH C18-SPE fraction inhibits chymotrypsin activity 98% (6 nmol pNA min^-1 mg^-1) and 99 % (4 nmol pNA min^-1 mg^-1), respectively. In contrast, trypsin activity was not inhibited by methanolic extracts of this cyanobacterium strain.

The synthesis of new collagen, chitosan and chitosan-collagen coated magnetic nanoparticles have been done. Two types of cross-linkers for polymer shell stabilization were used: glutaraldehyde (Gla) as a standard cross-linker and new one – squaric acid (SqA). Structure and morphology of prepared nanoparticles were characterized by ATR-FT IR, XRD and TEM analysis. The immobilization of lipase from Candida rugosa was performed on the nanoparticles surface. The amount of immobilized enzyme was quantified by the Bradford method. All of lipase-biopolymers coated nanoparticles were characterized with good activity recovery. A little hyperactivation of lipase immobilized on nanoparticles with SqA was observed. All of prepared lipase-immobilized nanoparticles were characterized with very good reusability.

Blocking the interaction of the SARS-CoV-2 spike protein with the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim in this study was to explore the efficacy of TCM treatments for SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. The antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis was found to be effective against SARS-CoV-2 infection through affecting the interaction of viral spike protein with the hACE2 protein. The active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside, respectively, found in S. baicalensis, exhibited strong inhibitory activities, targeting the viral spike protein or hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors of the interaction of the Omicron spike protein and hACE2 receptor from S. baicalensis. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed.

The aim of this study was to analyze other possible new markers of severity that can be assessed in patients with type 2 diabetes and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infection. Our study included 90 subjects: 45 patients with type 2 diabetes mellitus and infected with the SARS-CoV-2 virus, and 45 healthy persons as control. The serum level of ceruloplasmin at hospital admission was statistically significant directly correlated with the severity of COVID-19 (Coronavirus disease) (p=0.037) and with the serum level of IL-6 (Interleukine 6) (p=0.000). Ceruloplasmin was statistically significant inversely correlated with serum iron level (p=0.000). However, we observed that ACE (Angiotensin converting enzyme) decreased in severe forms of the infection with SARS-CoV-2 virus in patients with type 2 diabetes (p=0.001). Moreover, the decrease in ACE level was correlated with an increase in IL-6 level in these patients (p=0.001). IL-6 increase was statistically significant inversely correlated with serum iron, transferrin and ACE levels. There was a noticed a decreasing tendency of the transferrin depending on the severity of the COVID-19 infection (p=0.000). In addition to the known severity factors in the context of infection with the new coronavirus, decreased concentrations of ceruloplasmin, ACE and transferrin may represent new markers of COVID-19 severity in patients with type 2 diabetes. These parameters analyzed at the hospital admission could show the evolution towards the severe forms of infection with the SARS-CoV-2 virus.

Manipulation of the intestinal microbiome is an emerging area of research, especially in hind-gut fermenters such as the horse. The aim of this study was to determine whether fecal pH can provide a simple and cost-effective tool to document changes in the intestinal metabolome and microbiome following supplementation of feed with an enzyme rich malt extract (ERME). Fecal pH was determined weekly in triplicate using a commercial soil pH meter in 72 Thoroughbred racehorses in training before and during supplementation with ERME (300mls per day divided into two feeds) alongside their regular feeding schedule. No control group was included. Fecal pH increased over the 4 weeks of treatment from 6.20 (6.15-6.24) to 6.40 (6.38-6.44; P<0.0001). Changes were not observed at one-week intervals but were apparent after 2 weeks There was a significant increase in the proportion of horses with a ‘normal’ pH at the end of the study (55%) compared to the start of the study (11%; p<0.001). These data support the further validation of the use of fecal pH in large scale microbiome/metabolome studies in the horse, although comparisons with these outcomes are now warranted.

The polymerase chain reaction (PCR) is a widely used technique in the biosciences and has become increasingly popular in recent years. One of the key elements of this technique is the use of a DNA polymerase that is heat-stable and retains fidelity during the process. To this end, archaeal Fam-ily-B DNA polymerases are preferred due to their high thermostability and fidelity. In particular, the DNA polymerase from Thermus aquaticus (Taq DNApol) is widely utilized in PCR procedures. In this work, a novel in-silico structure-based methodology was employed to examine the most heat-tolerant DNA polymerase available. In spite of this, Thermococcus kodakarensis and Geobacillus stearothermophilus DNApol are more stable and heat-tolerant DNApols due to their high number of intra-protein interactions. Variations in the content of polar amino acids also played a significant role in the increase in heat stability. A further factor contributing to the stability of proteins is the stabilization of helix in secondary structure through the use of charged amino acids. DNApol from these organisms has been shown to be suitable for use in PCR, as well as in other biological processes able to withstand high temperatures. In this study, it has been demonstrated that im-provements in PCR performance can be easily obtained by blending elements from closely related archaeal polymerases, a strategy that may, in the future, be extended to other archaeal polymer-ases. This approach allowed for a comprehensive analysis of the enzyme's thermal stability and fidelity, leading to an improved understanding of the polymerase's properties and potential ap-plications

The paper focuses on the application of a multilevel, integrative and territorial approach to the protection of cultural heritage through planning instruments in Serbia. It points out the problems of the sectoral approach and discusses international recommendations that can be integrated into the planning methodology. The case study is the area of Djerdap National Park, which has an already established international and national importance. The research suggests that the cultural heritage in the area, with the exception of the Roman limes, is still recognized at the level of individual localities. The study emphasizes the importance of aligning all levels of governance to safeguard the whole spatial entity rather than focusing solely on its particular areas. The interna-tional and national importance of the planning area contributes to the strengthening of awareness of heritage and its institutional protection. However, the vitality of the area, which is key to its sustainable development, comes from the local level. Planning documents contribute to the inte-gration of local values and local communities into development programs and projects.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in the alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more response in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Background: Educating college students about the importance of good sleep habits could lead to the adoption of healthier sleep routines, including sufficient sleep duration and sleep quality. The current pre-post study examines the impact of completing a sleep education program called “Sleep 101” on (1) sleep hygiene practices, (2) sleep quality, and (3) weekday and weekend sleep behaviors among college students. “Sleep 101” is an interactive online course developed to educate college students about the importance of sleep by addressing basic sleep physiology, the impact of sleep on mood, academic and physical performance, the interactions between sleep and various substances, and common sleep disorders. Methods: 338 college students were recruited from a midsized public university in the US and invited to participate in “Sleep 101”, of which 25 participants completed the baseline and post-intervention questionnaire. The Sleep Hygiene Index (SHI) was utilized to evaluate sleep hygiene practices, while standard questions were employed to assess weekday and weekend sleep behaviors and overall sleep quality. Results: Those who completed the study demonstrated a significant increase in weekday sleep duration and were less likely to engage in negative prior bedtime activities. In addition, 40% of those who completed baseline and post-intervention assessments noticed improvements in their sleep quality. Conclusion: These findings suggest that a brief online program is beneficial in improving sleep behaviors among college and could be a scalable approach to addressing current sleep issues and preventing future sleep problems in college students.

The aim of this study was to investigate the potential of converting potato peel waste into bio ethanol using an in-house produced multi-enzyme preparation. To achieve this, a variety of pre-treatment strategies, including thermal, chemical, and thermo-chemical methods, were applied to the potato peels. These included boiling for 30 and 60 min, steam at varying pressures and times, and different concentrations of chemicals such as H2SO4, HNO3, CH3COOH, HCl, NaOH, Ca(OH)2, KOH, NH3, and H2O2, either alone or in combination with steam. The pre-treated potato peels were then enzymatically hydrolyzed using a crude multi-enzyme cocktail derived from solid-state fermentation of wheat bran by a natural variety of Aspergillus niger P-19, containing cellulases, hemicellulases, pectinase, and amylases. The most effective pre-treatment combination was found to be 3% H2SO4 followed by steam under pressure and enzymatic hydrolysis using the crude multi-enzyme preparation. This combination resulted in the highest yield of reducing sugars (141.04 ±12.31 g/l) with a 98.49% carbohydrate conversion at a 20% substrate loading, giving an ethanol yield of 43.2 g/l. Furthermore, supplementing the medium with peptone, (NH4)(H2PO4), and ZnSO4 at 0.1%w/v each with 22% and 24% solid, respectively, resulted in appreciable yields of 51.67 and 54.75 g/l, further enhancing the ethanol yield.