Esterase Activity Research Articles

Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer’s disease cortex

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer’s disease (AD). New MethodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Fluorescent Probe as Dual-Organelle Localizer Through Differential Proton Gradients Between Lipid Droplets and Mitochondria.

Dual-organelle molecular localizers represent powerful new tools allowing the exploration of interorganelle physical contacts and subcellular chemical communication. Here, we describe new dynamic molecular probes to localize mitochondria and lipid droplets taking advantage of the differential proton gradients present in these organelles as well as the activity of mitochondrial esterase. We unveil their potential utility when organelle retention mechanisms and proton gradients are synchronized, an insight that has not been documented previously. Our discoveries indicate that dual-organelle probes serve as a valuable multiplexing assay during starvation-induced autophagy. The pioneering molecular mechanism they employ opens doors to avoid using labile esters such as acetoxymethyl derivatives which are not optimal in imaging microscopy assays.

TRPA1 up-regulation mediates oxidative stress in a pulpitis model in vitro.

Pulpitis is associated with tooth hypersensitivity and results in pulpal damage. Thermosensitive transient receptor potential (TRP) ion channels expressed in the dental pulp may be key transducers of inflammation and nociception. We aimed at investigating the expression and role of thermo-TRPs in primary human dental pulp cells (hDPCs) in normal and inflammatory conditions. Inflammatory conditions were induced in hDPC cultures by applying polyinosinic:polycytidylic acid (poly(I:C)). Gene expression and pro-inflammatory cytokine release were measured by RT-qPCR and ELISA. Functions of TRPA1 channels were investigated by monitoring changes in intracellular Ca2+ concentration. Mitochondrial superoxide production was measured using a fluorescent substrate. Cellular viability was assessed by measuring the activity of mitochondrial dehydrogenases and cytoplasmic esterases. TRPA1 activity was modified by agonists, antagonists, and gene silencing. Transcripts of TRPV1, TRPV2, TRPV4, TRPC5, and TRPA1 were highly expressed in control hDPCs, whereas TRPV3, TRPM2, and TRPM3 expressions were much lower, and TRPM8 was not detected. Poly(I:C) markedly up-regulated TRPA1 but not other thermo-TRPs. TRPA1 agonist-induced Ca2+ signals were highly potentiated in inflammatory conditions. Poly(I:C)-treated cells displayed increased Ca2+ responses to H2O2, which was abolished by TRPA1 antagonists. Inflammatory conditions induced oxidative stress, stimulated mitochondrial superoxide production, resulted in mitochondrial damage, and decreased cellular viability of hDPCs. This inflammatory cellular damage was partly prevented by the co-application of TRPA1 antagonist or TRPA1 silencing. Pharmacological blockade of TRPA1 channels may be a promising therapeutic approach to alleviate pulpitis and inflammation-associated pulpal damage.

Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes

Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes

Efficient Enzymatic Synthesis of Carbamates in Water Using Promiscuous Esterases/Acyltransferases.

Biocatalysis provides an attractive approach to facilitate synthetic reactions in aqueous media. Motivated by the discovery of promiscuous aminolysis activity of esterases, we exploited the esterase fromPyrobaculum calidifontisVA1 (PestE) for the synthesis of carbamates from different aliphatic, aromatic, and arylaliphatic amines and a set of carbonates such as dimethyl-, dibenzyl-, or diallyl carbonate. Thus, aniline and benzylamine derivatives, aliphatic and even secondary amines could be efficiently converted into the correspondingbenzyloxycarbonyl(Cbz)- or allyloxycarbonyl (Alloc)-protected products in bulk water, with (isolated) yields of up to 99%.

Efficient Enzymatic Synthesis of Carbamates in Water Using Promiscuous Esterases/Acyltransferases

Biocatalysis provides an attractive approach to facilitate synthetic reactions in aqueous media. Motivated by the discovery of promiscuous aminolysis activity of esterases, we exploited the esterase from Pyrobaculum calidifontis VA1 (PestE) for the synthesis of carbamates from different aliphatic, aromatic, and arylaliphatic amines and a set of carbonates such as dimethyl‐, dibenzyl‐, or diallyl carbonate. Thus, aniline and benzylamine derivatives, aliphatic and even secondary amines could be efficiently converted into the corresponding benzyloxycarbonyl (Cbz)‐ or allyloxycarbonyl (Alloc)‐protected products in bulk water, with (isolated) yields of up to 99%.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

Dynamic interplay between soil microbial communities, enzyme activities, and pear quality across planting years

Few studies have been reported on the effect of planting years on fruit quality and soil of pear trees. In this study, four planting years (T5, T20, T30, and T40) of Cuiguan pears were used to analyze fruit quality, rhizosphere soil enzymes, and microbial diversity of pear trees, and their correlations. The results showed that the content of sucrose, reducing sugar and ascorbic acid in Cuiguan Pear showed a tendency of increasing and then decreasing with the increase of planting years, in which the highest content was found in 20- and 30-year-old fruits, and the highest content of total acid was found in 5-year-old fruits. Rhizosphere soil enzyme activities varied with planting year, with the highest protease activity in 20-year-old soil, phosphatase and urease in 30-year-old soil, polyphenol oxidase in 5-year-old soil, and sucrase in 40-year-old soil. The microbial diversity index and the number of OTUs showed an increasing and decreasing trend with the increase of planting years. Among the top 11 bacteria in pear rhizosphere soil average relative abundance, with Bradyrhizobium decreasing in relative abundance at the peak pear fruiting stages (T20 and T30), while Acidothermus showed an increasing trend in relative abundance with increasing planting years. RDA analysis showed that there were differences in the microbial community structure of pear trees at different planting years, and that both sucrose and reducing sugar contents in pears were positively correlated with T20 and T30, ascorbic acid content was positively correlated with T40, whereas the total acid content was positively correlated with T5, and that T20 was positively correlated with soil protease and phosphate mono esterase activities, and that T30 was positively correlated with polyphenol oxidase and urease activities, whereas T40 was positively correlated with sucrase activity. In summary, with the increase of planting years, changes in soil microbial community structure and soil enzyme activity have a significant impact on pear quality formation, and the results of the study provide a theoretical basis for scientific management of pear orchards.

Activity of insecticide detoxification enzymes in Pectinophora gossypiella (Saunders): Implications for insect pest management in cotton crops

The pink bollworm Pectinophora gossypiella is one of the most important endocarpic pests in cotton. In the management of this pest, the intensive use of synthetic insecticides has led to the reduction of its sensitivity to certain active ingredients. It is in this perspective that the study was conducted to determine the enzymes involved in the metabolism of insecticides in P. gossypiella. Insects were collected in five localities (Bouaké, Bouaflé, Vavoua, Sésuéla and Kounahiri) with high pest pressure in the southern cotton zone. The collected insects were ground for the determination of enzymes involved in insecticide metabolism. The analyses showed a significant increase in glutathione-δ-transferase activity in populations of Bouaké compared to the other localities. However, individuals from Séguéla expressed more esterase (alpha and beta) and oxidase activities. Alpha and beta esterase activities were also more expressed in Bouaflé individuals. The high enzymatic activity of esterases and oxidases in individuals from Séguéla and Bouaflé reflects a low sensitivity to insecticides in which esterases and oxidases are involved in the metabolism. On the other hand, in Bouaké, only glutathione- δtransferases are involved in the detoxification of insecticides.

­­­­A Bioorthogonal Dual Fluorogenic Probe for the Live‐Cell Monitoring of Nutrient Uptake by Mammalian Cells

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron‐Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein‐diacetate‐succinimidyl esters (CFSE) – a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity – with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron‐rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile‐containing nutrients in live primary immune cell populations using flow cytometry and live‐cell microscopy.

----A Bioorthogonal Dual Fluorogenic Probe for the Live-Cell Monitoring of Nutrient Uptake by Mammalian Cells.

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE) - a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity - with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.

Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

Deciphering the binding mechanisms of eugenol and 2-methylpyrazine with human serum albumin using technical approach

Eugenol (EGL) and 2-methylpyrazine (2-MTP) are common food additives that are used as flavors in a variety of foods. To study the effect of food additives on human serum albumin (HSA), it is necessary to deeply study the interaction of EGL and 2-MTP with HSA. Fluorescence, UV–vis absorption, circular dichroism (CD) and computational techniques showed EGL and 2-MTP bind statically to HSA, inducing conformational changes of HSA. We calculated the quenching constants (ksv), binding constants (kq), binding distances (r) and thermodynamic constants (ΔH0, ΔS0, ΔG0) at four different temperatures. Based on thermodynamic analysis and molecular dynamics (MD) simulations, the main forces of 2-MTP-HSA and EGL-HSA systems are hydrophobic interaction and π-π accumulation, respectively. In addition, the interaction alters the physiological activity of HSA, esterase activity and free radical scavenging ability. This study provides valuable insights into the molecular interactions of food additive compounds EGL and 2-MTP with HSA, the findings provide theoretical reference for the development of new food additives and drugs in the future.

Naringenin mitigates aluminum toxicity-induced learning memory impairments and neurodegeneration through amelioration of oxidative stress.

Aluminum chloride (AlCl3) is a potent neurotoxic substance known to cause memory impairment and oxidative stress-dependent neurodegeneration. Naringenin (NAR) is a dietary flavonoid with potent antioxidant and anti-inflammatory properties which was implemented against AlCl3-induced neurotoxicity to ascertain its neuroprotective efficacy. Experimental neurotoxicity in mice was induced by exposure of AlCl3 (10 mg/kg, p.o.) followed by treatment with NAR (10 mg/kg, p.o.) for a total of 63 days. Assessed the morphometric, learning memory dysfunction (novel object recognition, T- and Y-maze tests), neuronal oxidative stress, and histopathological alteration in different regions of the brain, mainly cortex, hippocampus, thalamus, and cerebellum. AlCl3 significantly suppressed the spatial learning and memory power which were notably improved by administration of NAR. The levels of oxidative stress parameters nitric oxide, advanced oxidation of protein products, protein carbonylation, lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, reduced glutathione, and the activity of acetylcholine esterase were altered 1.5-3 folds by AlCl3 significantly. Treatment of NAR remarkably restored the level of oxidative stress parameters and maintained the antioxidant defense system. AlCl3 suppressed the expression of neuronal proliferation marker NeuN that was restored by NAR treatment which may be a plausible mechanism. NAR showed therapeutic efficacy as a natural supplement against aluminum-intoxicated memory impairments and histopathological alteration through a mechanism involving an antioxidant defense system and neuronal proliferation.

Effect of microbial biostimulants on the antioxidant profile, antioxidant capacity and activity of enzymes influencing the quality level of raspberries (Rubus idaeus L.)

The influence of four microbial biostimulants containing various strains of Bacillus subtilis and/or Paenibacillus sp. on the quality of raspberries cv. Delniwa, Poemat, and Enrosadira cultivated in two consecutive seasons was investigated. The biostimulants influenced the antioxidant level, antioxidant capacity, phenolic acids and flavonoids profiles, enzymatic activity, and the degree of methylation and acetylation of the pectin in the raspberry fruits. The biostimulants had the greatest effect on the antioxidant content (16% - 20% increase) and capacity in the Delniwa raspberry fruits from the first season. A positive correlation was found between the activity of the β-galactosidase enzyme and ferric reducing power. In the second season, a decrease in the activity of pectin esterase and α-L-arabinofuranosidase and an increase in the degree of methylation of pectin were noted. Our results suggest that the changes in raspberry quality were related to the type of biostimulant applied.

Combined effects of three insecticides with different modes of action on biochemical responses of the solitary bee Osmia bicornis

Bees are simultaneously exposed to a variety of pesticides, which are often applied in mixtures and can cause lethal and sublethal effects. The combined effects of pesticides, however, are not measured in the current risk assessment schemes. Additionally, the sublethal effects of pesticides on a variety of physiological processes are poorly recognized in bees, especially in non-Apis solitary bees. In this study, we used a full-factorial design to examine the main and interactive effects of three insecticide formulations with different modes of action (Mospilan 20 SP, Sherpa 100 EC, and Dursban 480 EC) on bee biochemical processes. We measured acetylcholinesterase (AChE), glutathione S-transferase (GST) and esterase (EST) activities, as well as a nonenzymatic biomarker associated with energy metabolism, i.e., ATP level. All studied endpoints were affected by Sherpa 100 EC, and the activities of AChE and EST as well as ATP levels were affected by Dursban 480 EC. Moreover, complex interactions between all three insecticides affected ATP levels, showing outcomes that cannot be predicted when testing each insecticide separately. The results indicate that even if interactive effects are sometimes difficult to interpret, there is a need to study such interactions if laboratory-generated toxicity data are to be extrapolated to field conditions.

Assessment of species distribution and virulence factors of oral fungal carriage among hospitalized patients with COVID-19: a case-control study.

The COVID-19 pandemic highlighted the need to study oral fungal carriage and its potential impact. In oral fungal environments, factors like changes in respiratory epithelium, increased pathogen attachment, local inflammation, and virulence factors could influence COVID-19 severity. The authors conducted a study to explore oral fungal carriage in COVID-19 patients and compare it to a healthy control group. The authors executed a case-control investigation including 144 COVID-19 patients and an equivalent number of 144 healthy controls. The matching criteria encompassed age, sex, body mass index, and the history of antibiotic and antiviral medication intake. This research was performed over a span of 12 months from May 2021 to May 2022. The mouth area was sampled with a cotton-tipped swab. Subsequently, all the samples underwent fungal culture and PCR-sequencing procedures. In COVID-19 patients, oral fungal carriage was three times higher compared to healthy controls. Candida was the exclusive genus found in both groups, with Candida albicans being the most frequently isolated species (90.79%). Among COVID-19 patients, Candida species showed significantly higher esterase, proteinase, and hemolysin activity compared to healthy individuals. Both groups exhibited elevated levels of C. albicans virulence factors compared to non-albicans species. It is crucial to understand the way that virulence factors of oral fungal carriage act in COVID-19 patients in order to come up with novel antifungal medications, identify the contributing factors to drug resistance, and manage clinical outcomes.

Synergistic enzyme assisted release of ferulic acid from brewer´s spent grain

Brewer’s spent grain (BSG), the most abundant by-product of the beer industry, can be considered a sustainable source of ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA). FA is esterified to the arabinoxylan in the BSG. FA exhibits a variety of biological activities and is a target for valorization of BSG. We present a novel enzyme-assisted approach that utilizes the synergistic action of a specific bacterial endo-1,4-β-xylanase and a fungal ferulic acid esterase activity to selectively release high yields of FA from BSG. The study compares the efficacy of three different fungal feruloyl esterases (E.C. 3.1.1.73) of carbohydrate esterase family 1 (CE1), HiFae, AoFae1, and AnFae1, acting separately or in combination with two different endo-1,4-β-xylanases (EC 3.2.1.8), a bacterial GH30 enzyme from Dickeya chrysanthemi, and Shearzyme®, a commercial GH10 enzyme from Aspergillus aculeatus, respectively. On an equimolar dosage basis, the AnFae1-GH30 was the most efficient combination in releasing FA from BSG. This combination showed significant synergy in a dose-dependent manner, enabling up to 85% FA recovery from BSG within 30 min. of treatment. The results demonstrate a novel enzymatic approach for valorization of BSG as FA is considered a high-value natural compound for use in e.g. anti-aging skin care products.