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Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Background: Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge. Aim: This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns. Method: A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay. Results: The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes. Conclusion: The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans , can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing–dependent and RNA editing–independent mechanisms in regulating embryogenesis. [ABSTRACT FROM AUTHOR]

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8‐oxo‐dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism. [ABSTRACT FROM AUTHOR]

Objectives To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. Methods We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electronic medical records. Results PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. Conclusion Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement. [ABSTRACT FROM AUTHOR]

Olive tree anthracnose is caused by infection with Colletotrichum fungi, which in Portugal are mostly C. nymphaeae, C. godetiae, and C. gloeosporioides s.s. Severe economic losses are caused by this disease that would benefit from a greener and more efficient alternative to the present agrochemical methods. Yeasts are serious candidates for pre-harvest/in field biocontrol of fungal infections. This work identified the yeast Wickerhamomyces anomalus as a strong antagonizer of the three fungi and studied in vitro this ability and its associated mechanisms. Antagonism was shown to not depend on the secretion of volatile compounds (VOCs), or siderophores or any other agar-diffusible compound, including hydrolytic enzymes. Rather, it occurred mostly in a cell-to-cell contact dependent manner. This was devised through detailed microscopic assessment of yeast-fungus cocultures. This showed that W. anomalus antagonism of the three Colletotrichum proceeded through (i) the adhesion of yeast cells to the phytopathogen hyphae, (ii) the secretion of a viscous extracellular matrix, and (iii) the emptying of the hyphae. Yeasts ultimately putatively feed on hyphal contents, which is supported by light microscopy observation of MB and PI co-culture-stained samples. Accordingly, numerous W. anomalus cells were observed packing inside C. godetiae emptied hyphae. This behaviour can be considered microbial predation and classified as necrotrophic mycoparasitism, more explicitly in the case of C. godetiae. The results support the prospect of future application of W. anomalus as a living biofungicide/BCA in the preharvest control of olive anthracnose. [ABSTRACT FROM AUTHOR]

Context.--Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carba-penemase production, by evaluating carbapenem hydrolysis. Objective.--To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. Design.--We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene (blaKPC, blaGES, blaNDM-1, blaIMP, blaVIM, and blaOXA-48-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2-4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. Results.--Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For blaKPC-positive isolates, sensitivity was 97.9%, while it reduced significantly for blaNDM-1 and blaOXA-48-like isolates: 62.5% (10 of 16) and 66.7% (6 of 9), respectively. Indeed, logRQ was higher in blaKPC-positive isolates (0.371.97) than in blaNDM-1 (-1.37 to 0.83) and blaOXA-48-like isolates (-1.08 to 1.79). Conclusions.--This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures. [ABSTRACT FROM AUTHOR]

The aim of this study is to develop safe biological methods for controlling fungal deterioration of historical manuscripts. Therefore, fifteen fungal isolates were obtained from paper sheets and leather skins of a deteriorated historical manuscript (dated back to the 13th century). Those isolates were identified using both traditional methods and ITS-sequencing analysis. Aspergillus niger accounted for seven strains, Penicillium citrinum for one strain, Aspergillus flavus for three, Aspergillus fumigatus for one, Aspergillus nidulans for one, and Penicillium chrysogenum for two of the fungal strains that were obtained. The ability of fungal strains for the secretion of cellulase, amylase, gelatinase, and pectinase as hydrolytic enzymes was evaluated. The capability of the probiotic-bacterial strain Lactobacillus plantarum DSM 20174 for inhibition of fungal strains that cause severe deterioration was studied using ethyl acetate-extract. The metabolic profile of the ethyl acetate-extract showed the presence of both high- and low-molecular-weight active compounds as revealed by GC-MS analysis. The safe dose to prevent fungal growth was determined by testing the ethyl acetate extract's biocompatibility against Wi38 and HFB4 as normal cell lines. The extract was found to have a concentration-dependent cytotoxic impact on Wi38 and HFB4, with IC 50 values of 416 ± 4.5 and 349.7 ± 5.9 μg mL−1, respectively. It was suggested that 100 μg mL−1 as a safe concentration could be used for paper preservation. Whatman filter paper treated with ethyl acetate extract was used to cultivate the fungal strain Penicillium citrinum AX2. According to data analysis, fungal inhibition measurement, SEM, ATR-FT-IR, XRD, color change measurement, and mechanical property assessment, the recommended concentration of ethyl acetate extract was adequate to protect paper inoculated with the highest enzymatic producer fungi, P. citrinum AX2. [Display omitted] • Several fungal strains were obtained from old historical manuscripts. • The fungal strains were identified and investigated for enzymatic activities. • Probiotic L. plantarum DSM20174 metabolites were used to biocontrol fungal growth. • Ethyl acetate extract shows biocompatibility with Wi38 and HFB4 normal cell lines. • SEM, ATR-FT-IR, XRD, and mechanical properties recommended the extract application. [ABSTRACT FROM AUTHOR]

Mango is considered one of the most important tropical fruits worldwide in terms of its consumption and consumer acceptability. Its processing generates huge quantities of mango byproducts, which is often discarded unscrupulously into the environment and, therefore, needs effective waste management practices. The extraction of mango peels' dietary fiber using enzymatic method can be a useful valorization strategy for management of mango by-products. In the present investigation, dietary fiber (soluble and insoluble fraction) was extracted by enzymatic hydrolysis using α-amylase, protease, and amyloglucosidase. Highest yield of dietary fiber (67.5%, w/w) was obtained at 60 °C temperature using recommended enzyme concentrations including α-amylase (40 µL), protease (110 µL), and amyloglucosidase (200 µL) after a treatment time of 60 min. SEM analysis indicated the increased porosity of dietary fiber samples caused due to the hydrolytic effect of enzymes on its surface structure, whereas FTIR analysis confirmed the functional groups present in dietary fiber. The coexistence of crystalline and amorphous nature of polymers present in soluble and insoluble fractions of dietary fiber was assessed by XRD analysis. Further, the analysis of functional properties including WHC, OHC, and SC revealed the suitability of using extracted mango peel's dietary fiber in the food systems. [ABSTRACT FROM AUTHOR]

The genome of a cell is continuously battered by a plethora of exogenous and endogenous processes that can lead to damaged DNA. Repair mechanisms correct this damage most of the time, but failure to do so leaves mutations. Mutations do not occur in random manner, but rather typically follow a more or less specific pattern due to known or imputed mutational processes. Mutational signature analysis is the process by which the predominant mutational process can be inferred for a cancer and can be used in several contexts to study both the genesis of cancer and its response to therapy. Recent pan-cancer genomic efforts such as "The Cancer Genome Atlas" have identified numerous mutational signatures that can be categorized into single base substitutions, doublet base substitutions, or small insertions/deletions. Understanding these mutational signatures as they occur in non-small lung cancer could improve efforts at prevention, predict treatment response to personalized treatments, and guide the development of therapies targeting tumor evolution. For non-small cell lung cancer, several mutational signatures have been identified that correlate with exposures such as tobacco smoking and radon and can also reflect endogenous processes such as aging, APOBEC activity, and loss of mismatch repair. Herein, we provide an overview of the current knowledge of mutational signatures in non-small lung cancer. [ABSTRACT FROM AUTHOR]

The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (КА-1413, КА-342, КBl-29, КBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions. [ABSTRACT FROM AUTHOR]

Glycoside Hydrolase family 65 (GH65) is a unique family of carbohydrate-active enzymes. It is the first protein family to bring together glycoside hydrolases, glycoside phosphorylases and glycosyltransferases, thereby spanning a broad range of reaction types. These enzymes catalyze the hydrolysis, reversible phosphorolysis or synthesis of various α-glucosides, typically α-glucobioses or their derivatives. In this review, we present a comprehensive overview of the diverse reaction types and substrate specificities found in family GH65. We describe the determinants that control this remarkable diversity, as well as the applications of GH65 enzymes for carbohydrate synthesis. Key points: • GH65 is the first CAZy family to contain hydrolases, phosphorylases and transferases • Distinct residues and loops are determinants of substrate specificity in family GH65 • GH65 enzymes hold strong potential for carbohydrate synthesis via coupled reactions [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C‐terminal α‐helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D‐amino acid HADA, reporting on transpeptidase activity, compared to wild‐type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase‐mediated PG cleavage may be a rate‐limiting step for septal PG synthesis. [ABSTRACT FROM AUTHOR]

Biosensors are used to detect and quantify chemicals produced in industrial microbiology with high specificity, sensitivity, and portability. Most biosensors, however, are limited by the need for transcription factors engineered to recognize specific molecules. In this study, we overcome the limitations typically associated with traditional biosensors by engineering Pseudomonas putida for whole-cell sensing of a variety of chemicals. Our approach integrates fluorescent reporters with synthetic auxotrophies within central metabolism that can be complemented by target analytes in growth-coupled setups. This platform enables the detection of a wide array of structurally diverse chemicals under various conditions, including co-cultures of producer cell factories and sensor strains. We also demonstrate the applicability of this versatile biosensor platform for monitoring complex biochemical processes, including plastic degradation by either purified hydrolytic enzymes or engineered bacteria. This microbial system provides a rapid, sensitive, and readily adaptable tool for monitoring cell factory performance and for environmental analyzes. Biosensors are powerful tools for quantification of a wide range of molecules but require extensive engineering for each analyte. Here, the authors engineered a robust environmental bacterium for sensing a diverse set of chemicals, such as lactate and PET degradation products, via growth-coupling [ABSTRACT FROM AUTHOR]

Fungal phytopathogens represent a large and economically significant challenge to food production worldwide. Thus, the application of biocontrol agents can be an alternative. In the present study, we carried out biological, metabolomic, and genetic analyses of three endophytic isolates from nodules of Chamaecytisus albus, classified as Pseudomonas chlororaphis acting as antifungal agents. The efficiency of production of their diffusible and volatile antifungal compounds (VOCs) was verified in antagonistic assays with the use of soil-borne phytopathogens: B. cinerea, F. oxysporum, and S. sclerotiorum. Diffusible metabolites were identified using chromatographic and spectrometric analyses (HPTLC, GC-MS, and LC-MS/MS). The phzF, phzO, and prnC genes in the genomes of bacterial strains were confirmed by PCR. In turn, the plant growth promotion (PGP) properties (production of HCN, auxins, siderophores, and hydrolytic enzymes, phosphate solubilization) of pseudomonads were bioassayed. The data analysis showed that all tested strains have broad-range antifungal activity with varying degrees of antagonism. The most abundant bioactive compounds were phenazine derivatives: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, and diketopiperazine derivatives as well as ortho-dialkyl-aromatic acids, pyrrolnitrin, siderophores, and HCN. The results indicate that the tested P. chlororaphis isolates exhibit characteristics of biocontrol organisms; therefore, they have potential to be used in sustainable agriculture and as commercial postharvest fungicides to be used in fruits and vegetables. [ABSTRACT FROM AUTHOR]

Tyrosol and hydroxytyrosol are powerful phenolic antioxidants occurring in olive oil and in by-products from olive processing. Due to their high polarity, esterification or other lipophilization is necessary to make them compatible with lipid matrices. Hydroxytyrosol methyl carbonate is a more effective antioxidant than dibutylhydroxytoluene or α-tocopherol and together with tyrosol methyl carbonate exerts interesting pharmacological properties. The purpose of this work was the enzymatic preparation of alkyl carbonates of tyrosol and hydroxytyrosol. A set of 17 hydrolases was tested in the catalysis of tyrosol methoxycarbonylation in neat dimethyl carbonate to find an economically feasible alternative to the recently reported synthesis of methyl carbonates catalyzed by Novozym 435. Novozym 435 was, however, found to be the best performing catalyst, while Novozym 735, pig pancreatic lipase, lipase F-AK and Lipex 100T exhibited limited reactivity. No enzyme accepted 1,2-propylene carbonate as the acylation donor. Under optimized reaction conditions, Novozym 435 was used in the batch preparation of tyrosol methyl carbonate and hydroxytyrosol methyl carbonate in quantitative yields. The enzymatic methoxycarbonylation of tyrosol and hydroxytyrosol can also be used as a method for their selective protection in enzymatic syntheses of phenylethanoid glycosides catalyzed with enzymes comprising high levels of acetyl esterase side activity. [ABSTRACT FROM AUTHOR]

Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide. F. verticillioides produces highly toxic mycotoxin Fumonisin B1 (FB1) in infested commodities which makes these food and feeds unsafe for humans and animals. For pathogenic fungi to successfully penetrate its plant hosts, the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer. However, there is limited information on how cutinases transcriptionally regulated to impact F. verticillioides pathogenicity. In this study, our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F. verticillioides pathogenicity and FB1 biosynthesis. Gene deletion of cutinase transcription factor FvCTF1α did not affect the growth and morphology of the fungal mycelia on CMII medium, whereas the conidiation, utilization of sodium acetate and sodium oleate, stress tolerance against cell wall interfering agent, and the cutinase and pectinase activities in the ΔFvctf1α mutant were negatively impacted. FvCtf1α regulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters. In addition, FvCtf1α, containing a novel function in regulating FB1, interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6, resulting in decreased production of FB1 in the ΔFvctf1α strain. ΔFvctf1α exhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F. verticillioides. We also demonstrated that FvCtf1α regulated FvCUT3 and FvCUT4 differently; FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB, a homologous protein of FvCtf1α. Moreover, RNA-seq analysis showed that FvCtf1α was associated with many pathways, such as fatty acid metabolism, carbon source utilization, cell wall integrity, oxidative stress, and fumonisin synthesis in F. verticillioides. Our study demonstrated that FvCtf1α was not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F. verticillioides virulence and mycotoxin biosynthesis. [ABSTRACT FROM AUTHOR]

Cannabinol (CBN) is a secondary metabolite of cannabis whose beneficial activity on inflammatory diseases of human skin has attracted increasing attention. Here, we sought to investigate the possible modulation by CBN of the major elements of the endocannabinoid system (ECS), in both normal and lipopolysaccharide‐inflamed human keratinocytes (HaCaT cells). CBN was found to increase the expression of cannabinoid receptor 1 (CB1) at gene level and that of vanilloid receptor 1 (TRPV1) at protein level, as well as their functional activity. In addition, CBN modulated the metabolism of anandamide (AEA) and 2‐arachidonoylglicerol (2‐AG), by increasing the activities of N‐acyl phosphatidylethanolamines‐specific phospholipase D (NAPE‐PLD) and fatty acid amide hydrolase (FAAH)—the biosynthetic and degradative enzyme of AEA—and that of monoacylglycerol lipase (MAGL), the hydrolytic enzyme of 2‐AG. CBN also affected keratinocyte inflammation by reducing the release of pro‐inflammatory interleukin (IL)‐8, IL‐12, and IL‐31 and increasing the release of anti‐inflammatory IL‐10. Of note, the release of IL‐31 was mediated by TRPV1. Finally, the mitogen‐activated protein kinases (MAPK) signaling pathway was investigated in inflamed keratinocytes, demonstrating a specific modulation of glycogen synthase kinase 3β (GSK3β) upon treatment with CBN, in the presence or not of distinct ECS‐directed drugs. Overall, these results demonstrate that CBN modulates distinct ECS elements and exerts anti‐inflammatory effects—remarkably via TRPV1—in human keratinocytes, thus holding potential for both therapeutic and cosmetic purposes. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION To date, cider production has primarily relied on Saccharomyces cerevisiae. Introducing novel non‐Saccharomyces yeasts can enhance the diversity of cider properties. Among these, the Metschnikowia genus stands out for its ability to produce hydrolytic enzymes that may impact the sensorial and technological properties of cider. This study focused on evaluating the impact of three Metschnikowia species – Metschnikowia koreensis (Mk), M. reukaufii (Mr), and M. pulcherrima (Mp) – which exhibit acid protease and esterase activity, on the quality enhancement of cider.The research findings indicate that the overall quality of cider produced through co‐fermentation with these species surpassed that of cider fermented with mono‐fermentation of S. cerevisiae (Sc). The cider fermented with the Sc + Mk combination exhibited the lowest levels of harsh‐tasting malic acid and higher levels of softer lactic acid. Sensory array analysis also demonstrated that the Sc + Mk fermented cider exhibited high sensor response values for compounds contributing to a complex overall olfactory composition and richness. Furthermore, the Sc + Mk fermented cider exhibited the highest total quantity and variety of volatile organic compounds (VOCs). Specifically, the concentrations of phenethyl alcohol, 3‐methyl‐1‐butanol, ethyl octanoate, and decanoic acid were notably elevated in comparison with other groups.This study illustrates that Metschnikowia species, particularly M. koreensis, show significant potential as starters for cider due to their various technological properties, including acidity modulation, aroma enhancement, and color improvement. The findings of this study provide a foundation for improving cider quality by co‐fermenting S. cerevisiae with innovative starter cultures. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

AbstractThis study explores a novel enzymatic pretreatment approach in anaerobic reactors for dairy wastewater, using lipase AY Amano to enhance methane production and modify microbial and archaeal community composition. Batch and semi-batch reactors with a total volume of 2000 mL were used to treat dairy wastewater with initial COD of 2000 and 15,000 mg L−1, respectively. In a new novel approach, the semi-batch reactors underwent a three-phase operation: 30 days of acclimation, 30 days of rest, and 30 days of active operation. Adding lipase (0.05% wv−1) as a pretreatment significantly increased methane yield over the 90 days by 135–138% compared with the control (without enzyme addition). The organic loading rate reached 0.22 g COD day−1 L−1. Furthermore, 30 days after the end of the semi-batch reactor approach (120 days from the start), reusing sludge in batch reactors increased methane yield by 114–122% compared to the control. This increase was linked to the emergence and shift of new methanogenic communities within the sludge. Integrating hydrolytic enzymes into the anaerobic treatment enhances performance and sustainability by fostering methanogen-enriched microbial communities. This is crucial for maximizing methane production but may increase costs, requiring further economic feasibility research. [ABSTRACT FROM AUTHOR]

We present a novel tool designed for the semiautomatic detection and assessment of prostate cancer during recurrence diagnostics using PET-CT with prostate-specific membrane antigen (PSMA). The tool automatically generates a structured report, which is enhanced by 3D images and ensures the reproducibility of the findings. Moreover, the area of the findings is automatically determined and can be corrected manually. Image data from 50 patients following radical prostatectomy was used. Our approach and its user interface were qualitatively evaluated by a nuclear medicine specialist. The segmentation analysis revealed a requirement for further automation. The resulting report was deemed highly beneficial, particularly in terms of the resulting reproducibility and the presentation of 3D images depicting the findings. The presented tool has great potential for much-needed support in everyday clinical practice, as it facilitates the communication of the diagnostic results to colleagues and patients and thus prevents misunderstandings. [ABSTRACT FROM AUTHOR]

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation. The degradation of inner autophagosomal membranes remains poorly understood. Here, Makar et al. show that RNAseK is involved in a lysosomal hydrolase delivery pathway that contributes to autophagosomal membrane degradation. [ABSTRACT FROM AUTHOR]

The unіque specіfіcіty of enzymes makes bіocatalytіc processes more technologіcally advanced compared to chemіcal ones. Hydrolytіc enzymes are the most wіdely used. Currently, enzyme preparatіons (EP) based on hydrolytіc enzymes are іncreasіngly used іn pharmaceutіcal productіon, medіcіne, food and chemіcal іndustrіes. The practіcal use of proteolytіc enzymes іs varіed, the most sіgnіfіcant іs theіr use іn surgery for the treatment of wound surfaces of varіous etіologіes, whіch іs due to the localіty of actіon: the rapіd rejectіon of non-vіable tіssue, wіthout affectіng healthy areas. The goal of the work іs to develop stabіlіzed forms of hydrolytіc enzymes that can be used for technіcal and pharmaceutіcal purposes, as well as to evaluate theіr effectіveness based on the analysіs of theіr kіnetіc and thermodynamіc parameters. [ABSTRACT FROM AUTHOR]

To investigate the effects of intercropping faba beans on soil fertility and microbial communities in pear orchards, soil samples were collected from no intercropping (CK), intercropped Sucan 3 Hao (CT1), intercropped Sucan 4 Hao (CT2), and intercropped Cican 1 Hao (CT3, local variety) treatments during the seedling stage, flowering stage, and maturity stage. The samples were analyzed for soil physicochemical properties, microbial activity, and community structures. The results showed that after intercropping with faba beans, the nitrate nitrogen (NO-3-N), and available phosphorus (Olsen P) increased during the seedling stage. During the flowering stage, soil electrical conductivity, dissolved organic carbon (DOC), dissolved organic nitrogen (DON), and NO-3-N contents significantly increased, while pH and Olsen P contents significantly decreased. Intercropping with faba beans increased soil microbial biomass carbon (MBC), especially CT1 and CT2 increased by 36% and 51%, respectively at the maturity stage compared with CK (P<0.05), and decreased the activity of hydrolytic enzymes related to the degradation of carbon, nitrogen, and phosphorus. Additionally, intercropping with faba beans increased the bacterial richness index and the Shannon index. It also increased the relative abundances of bacteria from the Planctomycetota, Firmicutes, Gemmatimonadetes, and Verrucomicrobia phyla, while decreasing the relative abundances of bacteria from the Proteobacteria, Actinobacteria, and Acidobacteria phyla. Mantel and CCA analyses indicated that pH and NO-3-N were important driving factors for microbial activity, and MBC significantly affected the bacterial community structures. Our results indicates that longterm intercropping of faba beans helps to increase soil microbial biomass and diversity, playing a crucial role in maintaining soil microecological diversity. [ABSTRACT FROM AUTHOR]

Mucin glycoproteins are a significant source of carbon for the gut bacteria. Various gut microbial species possess diverse hydrolytic enzymes and catabolic pathways for breaking down mucin glycans, resulting in competition for the limited nutrients within the gut environment. Adherence to mucin glycans represents a crucial strategy used by gut microbes to access nutrient reservoirs. Understanding these properties is pivotal for comprehending the survival mechanisms of bacteria in the gastrointestinal tract. However, characterization of individual strains within the vast array of coexisting bacteria in the microbiome is challenging. To investigate this, we developed mucin-immobilized particles by immobilizing porcine gastric mucin (PGM) onto glass beads chemically modified with boronic acid. These PGM-immobilized particles were then anaerobically cultured with human fecal microbiota, and the bacteria adhering to PGM were isolated. Interestingly, the microbiome composition remained largely unchanged irrespective of PGM immobilization. Nonetheless, bacteria isolated from PGM-immobilized glass particles exhibited notably higher N-acetylgalactosaminidase activity compared to the control beads. Furthermore, Bacteroides strains isolated from PGM-immobilized glass particles displayed enhanced adhesive and metabolic properties to PGM. These findings underscore the utility of PGM particles in enriching and isolating specific microbes. Moreover, they highlight substantial differences in microbial properties at the strain level. We anticipate that PGM-immobilized particles will advance culture-based microbiome research, emphasizing the significance of strain-level characterization. [ABSTRACT FROM AUTHOR]

Mutations that cause loss of function of GlcNAc-1- phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4 Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to fulllength PTase. In this dimeric structure, the EF-hand of one protomer is observed interacting with the conserved region four of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases. [ABSTRACT FROM AUTHOR]

Lead (Pb) is one of the most common environmental pollutants. Lead has an acute toxic effect on soil biotas and the enzymatic system of soils. The objective of this study is to carry out enzymatic diagnostics of soil health in the European part of Russia after Pb contamination. As a part of the simulation experiment, Pb (at maximum permissible concentrations (MPCs) of 1, 10, and 100) was used to contaminate 12 types of soils in the south and center of the European part of Russia, which differed in their physical and chemical properties. To assess soil health, the activity of oxidoreductases (catalase, dehydrogenases, and cysteine reductase) and hydrolases (invertase, urease, and phosphatase) was studied. Most enzymes were inhibited with increased Pb dosage. The most sensitive soils to Pb contamination, assessed by enzyme activity, are soils of semi-deserts and dry steppes. Cysteine reductase is considered the most sensitive enzyme to Pb contamination. The most informative indicators for Pb contamination were phosphatase, cysteine reductase, and invertase. The P (phosphatase) cycle and the redox enzyme (catalase) also have instability in Pb-contaminated soils. The C (invertase and dehydrogenases) and N (urease) cycles do not change significantly when contaminated with lead. The results of this study can be used for the diagnostics of the condition of soils in different natural areas after Pb contamination. [ABSTRACT FROM AUTHOR]

The Candida haemulonii complex includes emerging opportunistic human fungal pathogens with documented multidrug-resistance profiles. It comprises Candida haemulonii sensu stricto, Candida haemulonii var. vulnera, Candida duobushaemulonii, Candida pseudohaemulonii, and Candida vulturna. In recent years, rates of clinical isolation of strains from this complex have increased in multiple countries, including China, Malaysia, and Brazil. Biofilm formation, hydrolytic enzymes, surface interaction properties, phenotype switching and cell aggregation abilities, extracellular vesicles production, stress response, and immune evasion help these fungi to infect the host and exert pathological effects. Multidrug resistance profiles also enhance the threat they pose; they exhibit low susceptibility to echinocandins and azoles and an intrinsic resistance to amphotericin B (AMB), the first fungal-specific antibiotic. AMB is commonly employed in antifungal treatments, and it acts via several known mechanisms. Given the propensity of clinical Candida species to initiate bloodstream infections, clarifying how C. haemulonii resists AMB is of critical clinical importance. This review outlines our present understanding of the C. haemulonii complex's virulence factors, the mechanisms of action of AMB, and the mechanisms underlying AMB resistance. [ABSTRACT FROM AUTHOR]

Vulvovaginal candidiasis is considered one of the most common women's infections. The main cause of this disease is Candida albicans, which have many virulence factors such as germ tube formation, chlamydospore, and many hydrolytic enzymes. The current study aims to use anti-mannan antibodies and TLR9 to diagnose candidiasis in immuno-compromised patients with vulvovaginitis. A total of two hundred samples were obtained from patients attending Kirkuk Tumor Center, Kirkuk, Iraq, of which 100 were vaginal swabs from immunocompromised patients aged between (18-<40 years) from the period November 2022 to March 2023. The swab samples were transported with brain-hart infusion broth (Himedia-India). Furthermore, the other 100 samples were blood samples that were separated to use the serum for the detection of TLR9 and anti-mannan antibodies. The findings indicated that Candida spp. was present in 83% of the cultured samples. A notable rise in TLR9 was observed in serum samples that tested positive for candida spp. The results of the sensitivity and specification of IgM detection using t he ELISA test showed 100% and 89.4%, respectively. The same test for the detection of IgG showed 92.4% and 51.1%. The positive and negative agreement between the ELISA test for detecting IgM and the ELISA test for detecting IgG is 81.4% and 44.1%, respectively. The findings suggest that the use of anti-mannan antibodies to diagnose candidiasis should be considered and given more importance for identification purposes. [ABSTRACT FROM AUTHOR]

Sponge microbiomes are often highly diverse making it difficult to determine which lineages are important for maintaining host health and homeostasis. Characterising genomic traits associated with symbiosis can improve our knowledge of which lineages have adapted to their host and what functions they might provide. Here we examined five microbial families associated with sponges that have previously shown evidence of cophylogeny, including Endozoicomonadaceae, Nitrosopumilaceae, Spirochaetaceae, Microtrichaceae and Thermoanaerobaculaceae, to better understand the mechanisms behind their symbiosis. We compared sponge‐associated genomes to genomes found in other environments and found that sponge‐specific clades were enriched in genes encoding many known mechanisms for symbiont survival, such as avoiding phagocytosis and defence against foreign genetic elements. We expand on previous knowledge to show that glycosyl hydrolases with sulfatases and sulfotransferases likely form multienzyme degradation pathways to break and remodel sulfated polysaccharides and reveal an enrichment in superoxide dismutase that may prevent damage from free oxygen radicals produced by the host. Finally, we identified novel traits in sponge‐associated symbionts, such as urea metabolism in Spirochaetaceae which was previously shown to be rare in the phylum Spirochaetota. These results identify putative mechanisms by which symbionts have adapted to living in association with sponges. [ABSTRACT FROM AUTHOR]

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus , and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma. [ABSTRACT FROM AUTHOR]

Bile acid metabolism is partially regulated through the activity of the gut microbiota. Primary bile acids can be deconjugated and modified by bacteria expressing bile salt hydrolase and other enzymes, changing bile acid recycling by changing the interactions between enterocytes and hepatocytes. The modified bile acids can also activate signalling in cells regulating metabolism including colonic L-cells, skeletal muscle cells and brown adipocytes. In pregnancy, both bile acid metabolism and gut microbiota composition are altered. In women with intrahepatic cholestasis of pregnancy, the changes in bile acid metabolism are exacerbated and there is some evidence that the gut microbiota composition is also altered. Here we review the crosstalk between the liver and the gut especially in women with intrahepatic cholestasis of pregnancy, with a focus on the role of the gut microbiota in this crosstalk. [ABSTRACT FROM AUTHOR]

Adenosine deaminase 2 (ADA2) deficiency is an autosomal recessively inherited autoinflammatory disorder caused by loss-of-function mutations in the ADA2 gene. Although the pathogenesis involves the triggering of a proinflammatory cascade due to increased production of inflammatory cytokines such as tumor necrosis factor (TNF)-α and dysregulation of neutrophil extracellular trap formation resulting from an excess accumulation of extracellular adenosine, the pathogenetic mechanism still needs further clarification due to the broad clinical spectrum. In addition to the initially described vasculitis-related symptoms, hematological, immunological, and autoinflammatory symptoms are now well recognized. The diagnosis is made by demonstration of pathogenic variants of ADA2 with biallelic loss of function and identification of low plasma ADA2 catalytic activity. Currently, TNF-α inhibitors are the treatment of choice for controlling vasculitis manifestations and preventing strokes. However, in patients presenting with severe hematologic findings, TNF-α inhibitors are not the treatment of choice and hematopoietic stem cell transplantation has been shown to be successful in selected cases. Recombinant ADA2 protein and gene therapy are promising treatment modalities for the future. In conclusion, ADA2 deficiency has a broad phenotype and should be considered in the differential diagnosis of different clinical situations. In this review, we summarize the disease manifestations of ADA2 deficiency and available treatment options. [ABSTRACT FROM AUTHOR]

Objective: The study aimed to assess Sirt6 levels in serum of knee osteoarthritis (OA) patients compared to healthy individuals to evaluate its correlation with OA and to understand how Sirt6 is linked with the change in IL-6 levels. Methods: The cross-sectional study involved 50 knee OA patients clinically diagnosed as per the American College of Rheumatology guidelines and 50 healthy controls. Radiological examination as per Kellgren–Lawrence (KL) criteria was done to determine the disease severity. Peripheral blood samples were collected from each participant, and serum Sirt6 and IL-6 levels were measured using ELISA. Results: The serum Sirt6 levels in knee OA patients were significantly lower as compared to healthy controls (p = 0.023). Patients with knee OA of KL grade 4 had significantly lower Sirt6 levels as compared to those with KL grade 2 OA (p = 0.031). Individuals of younger age group had higher Sirt6 levels compared to older age group. IL-6 levels in knee OA patients were significantly higher as compared to controls (p = 0.007). A negative correlation was observed between serum Sirt6 and IL-6 levels (r = − 0.407; p = 0.035). Conclusion: The study concludes that serum Sirt6 levels are inversely associated with knee OA and may serve as a potential biomarker for the disease. Moreover, a negative correlation between Sirt6 and IL-6 levels was observed in this study. Further investigations are necessary to confirm these findings and to explore the mechanisms by which Sirt6 and IL-6 are involved in OA. [ABSTRACT FROM AUTHOR]

Background: Today, biologically active additives, which contain hydrolytic enzymes (amylase, lipase, protease), are widely used in the digestive industry. These enzymes are often obtained from animal and vegetable raw materials, -for example, from the pancreas of a reindeer, the Fabricius bag of chickens, and Medusomyces Gisevii Lindau (Kombucha). However, in obtaining enzymes, the activity of amylase and lipase may decrease due to active forms --of proteases. Objective: The main goal of this research work was to evaluate the stabilizing effect of nanostructures-cyclodextrins on the activity of hydrolytic enzymes (protease, amylase, lipase) obtained from animal raw material (chicken bursa of Fabricius) and microorganism cultures (Kombucha). Identify the relationship at which the negative influence of external environmental factors is leveled and, as a result, enzymatic activity is maintained for a certain period of time. Methods: The protein concentration in the solution was determined using the Lowry method. A modified method by Rukhlyadiva was used to determine the amylolytic activity. The definition of lipolytic activity is based on modified Shibabi-Bishop method. To determine the amylolytic activity, a modified method by Rukhlyadiva was used. Results: The relationship between b-cyclodextrin and enzymes was determined, at which enzymatic activity is maintained for a certain period. Conclusion: Thus, cyclodextrins in solution stabilizes the activity of proteases, amylases, and lipases, which is of great practical importance in the food and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Acetaminophen (APAP) overdose can induce hepatocyte necrosis and acute liver failure in experimental rodents and humans. APAP is mainly metabolized via hepatic cytochrome P450 enzymes to generate the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which forms acetaminophen protein adducts (APAP-adducts) and damages mitochondria, triggering necrosis. APAP-adducts and damaged mitochondria can be selectively removed by autophagy. Increasing evidence implies that the activation of autophagy may be beneficial for APAP-induced liver injury (AILI). In this minireview, we briefly summarize recent progress on autophagy, in particular, the pharmacological targeting of SQSTM1/p62 and TFEB in AILI. [ABSTRACT FROM AUTHOR]

Herein, we propose an analytical approach based on intermolecular fluorescent resonant energy transfer (FRET) pairs for the visualization of specific enzyme activity in model biomembranes and in living cells. Cell visualizations with fluorescent confocal laser microscopy usually rely on fluorescent probes, such as Fluorescein isothiocyanate (FITC), Alexa488, Tetramethylrhodamine isothiocyanate (TRITC) and many others. However, for more specific tasks, such as the detection of certain enzymatic activity inside the living cell, the toolbox is quite limited. In the case of enzyme-hydrolases for example, the choice is limited to organic molecules comprising a fluorescent dye (typically, 4-methylumbelliferone (MUmb) or 7-amino-4-methylcoumarin (AMC) derivatives) and a fluorescence quencher, bound via an enzyme-sensitive linker—so that when the linker is degraded, the fluorescent signal increases. Unfortunately, both MUmb and AMC are quenched and have a relatively low quantum yield in cells, and their excitation and emission ranges overlap with that of intracellular fluorophores, often producing a strong background noise. R6G, on the other hand, has excellent quantum yield apart from intracellular fluorophores, but there are no efficient quenchers that could be chemically linked to R6G. Herein, we show that R6G is able to form intermolecular FRET pairs with MUmb or AMC, with the latter serving as fluorescence donors. This yields a combination of R6G's excellent fluorescence properties with a possibility to use an enzyme-sensitive linker in MUTMAC or AMC derivatives. This phenomenon was initially discovered in a model system, reversed micelles, where the donor, the acceptor, and the enzyme are forced to be in close proximity to each other, so that proximity could serve as an explanation for the intermolecular FRET effect. Surprisingly enough, the phenomenon has been reproduced in living cells. Moreover, we were able to create working intermolecular donor–acceptor FRET pairs for several different enzymes, including chymotrypsin, phosphatase, and asparaginase. This appears counterintuitive, as besides the overlap of the emission spectra of the donor and the absorption spectra of the acceptor, there are other criteria for the FRET effect, including the convergence of two fluorophores at a distance of about 1–10 nm, and the orientation of their dipoles at a certain angle, which is difficult to imagine in a bulk system like a living cell. We hypothesize that FRET-enabling donor–acceptor interaction may be taking place at the inner surface of the lipid bilayer, to which both donor and acceptor molecules would likely have an affinity. This hypothesis would require a more detailed investigation. Therefore, we have shown that the method suggested has good potential in the visualization of enzyme functioning inside living cells, which is often a challenging task. Shifting of the fluorescence signal to the long-wavelength region would increase the signal selectivity, making it easily distinguishable from autofluorescence. [ABSTRACT FROM AUTHOR]

The current investigation was conducted with the objective of identifying yeasts that possess the ability to secrete a range of hydrolytic enzymes. Yeast isolates were collected from various samples, and the morphological characteristics of both the colony and yeast cells were examined. The capacity for enzyme production was assessed in multiple isolates by inoculating them in different media and quantifying their activities in terms of pectinase, amylase, urease, lipase, β-galactosidase, and protease. Out of a total of 42 yeast isolates, 30 displayed enzymatic activity. It is plausible that the yeast isolates obtained in this study hold potential for the production of industrial enzymes. Molecular identification techniques were employed to identify two isolates that exhibited multi-enzyme production and the highest enzyme activities. It was determined that these isolates belonged to two genera of yeasts, namely Pichia and Hanseniaspora. Specifically, isolate Y 16 was identified as Pichia kudriavizvii OL621856, while isolate Y 26 was identified as Hanseniaspora guillermondii OL621857 strains. [ABSTRACT FROM AUTHOR]

Objective To review how different classes of immunosuppressants affect wound healing. Data Sources A literature search was conducted in PubMed, Google Scholar, and the University of Calgary Health Sciences Library. Study Selection The researchers initially screened article titles using key words such as "immunosuppressive medication," "wound healing," and "immunosuppression." Articles in which the title and/or abstract contained these key words, that addressed wound healing related to immunosuppressant medications, and were published after 2000 were included in the review. When human data were not available for an immunosuppressant (class), animal studies were included. Data Extraction The 61 included articles underwent full text review and summarization. Data Synthesis: All included studies were summarized descriptively including immunosuppressive mechanism of action, study participants or subjects, and evidence of effects on wound healing. Conclusions Corticosteroids and mechanistic target of rapamycin inhibitors most consistently demonstrate detrimental effects on wound healing. For other classes of immunosuppressants, evidence is limited with varying effects on wound healing described. Larger, high-quality studies are required to better understand the effects of immunosuppressants, including those with new mechanisms of action, to identify those with the most impact on wound healing. [ABSTRACT FROM AUTHOR]

Sawdust, a major waste product of the forestry industry, is accumulating along the Lagos Lagoon in Lagos, Nigeria, without it being effectively managed. Besides its use in manufacturing sound-absorbing boards to reinforce concrete beams and for energy purposes, its potential as a renewable energy source and feedstock for bio-product development has not yet been realized. Cellulose, a glucose biopolymer and structural component of cellulose can be hydrolyzed by a hydrolytic enzyme known as cellulase. During the process, the enzyme breaks the B-1,4-glucosidic bond, which keeps the glucose units together, and by acting on this bond, numerous glucose units are released. As part of sawdust, the cellulose molecule is not freely available for the degradation action of the cellulase enzyme as it is strongly associated with lignin, which acts as bio-glue, keeping cellulose and hemicellulose together. Delignification is an effective technique that was used to make the sawdust from ten different trees along the Lagos Lagoon in Nigeria more susceptible to saccharification by cellulase isolated from the fungus Aspergillus niger. Delignified and non-delignified sawdust masses between 2 mg and 10 mg were incubated with the A. niger cellulase solution (2 mg.mL-1), whereafter, the amount of sugar produced by the cellulase action was determined. The percentage saccharification of each sawdust material was also linked with the amount of sugar produced during cellulase action. From these investigations was concluded that delignification increased sugar production when almost all the masses of different sawdust materials were degraded. It was also observed that the ratio of sawdust mass to enzyme concentration is an important variable that influences the effectiveness of the saccharification process. The percentage saccharification of the various sawdust materials was also determined, and it indicated that the highest percentage of saccharification was not obtained when the highest amount of sawdust was degraded, producing the highest amount of sugar. The saccharification of sawdust could contribute to the development of renewable energy sources and feedstock for bioproduct development. The process is, however, not that straightforward as variables such as the type of cellulase enzyme, pretreatment of the cellulose substrate, and optimizing of cellulase to cellulose ratio are a few that need to be optimized for the process to be effective in terms of glucose production. [ABSTRACT FROM AUTHOR]

The accelerating spread of antibiotic resistance has significantly weakened the clinical efficacy of existing antibiotics, posing a severe threat to public health. There is an urgent need to develop novel antimicrobial alternatives that can bypass the mechanisms of antibiotic resistance and effectively kill multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are one of the most promising candidates to treat MDR pathogenic infections since they display broad-spectrum antimicrobial activities and are less prone to achieve drug resistance. In this study, we investigated the antibacterial capability and mechanisms of two machine learning-driven linear peptide compounds termed YI12 and FK13. We reveal that YI12 and FK13 exhibit broad-spectrum antibacterial properties against clinically significant bacterial pathogens, inducing no or minimal hemolysis in mammalian red blood cells. We further ascertain that YI12 and FK13 are resilient to heat and acid-base conditions, and exhibit susceptibility to hydrolytic enzymes and divalent cations under physiological conditions. Initial mechanistic investigations reveal that YI12 and FK13 compromise bacterial membrane integrity, leading to membrane potential dissipation and excessive reactive oxygen species (ROS) generation. Collectively, our findings highlight the prospective utility of these two cationic amphiphilic peptides as broad-spectrum antibacterial agents. [ABSTRACT FROM AUTHOR]

Wildfire is a disturbance expected to increase in frequency and severity, changes that may impact carbon (C) dynamics in the soil ecosystem. Fire changes the types of C sources available to soil microbes, increasing pyrogenic C and coarse downed wood, and if there is substantial tree mortality, decreasing C from root exudates and leaf litter. To investigate the impact of this shift in the composition of C resources on microbial processes driving C cycling, we examined microbial activity in soil sampled from an Oregon burn 1 year after fire from sites spanning a range in soil burn severity from unburned to highly burned. We found evidence that postfire rhizosphere priming loss may reduce soil C loss after fire. We measured the potential activity of C‐acquiring and nitrogen (N)‐acquiring extracellular enzymes and contextualized the microbial resource demand using measurements of mineralizable C and N. Subsurface mineralizable C and N were unaltered by fire and negatively correlated with hydrolytic extracellular enzyme activity (EEA) in unburned, but not burned sites. EEA was lower in burned sites by up to 46%, but only at depths below 5 cm, and with greater decreases in sites with high soil burn severity. These results are consistent with a subsurface mechanism driven by tree mortality. We infer that in sites with high tree mortality, subsurface EEAs decreased due to loss of rhizosphere priming and that inputs of dead roots contributed to mineralizable C stabilization. [ABSTRACT FROM AUTHOR]

The ever-increasing presence of micropollutants necessitates the development of environmentally friendly bioremediation strategies. Inspired by the remarkable versatility and potent catalytic activities of microbial enzymes, researchers are exploring their application as biocatalysts for innovative environmental cleanup solutions. Microbial enzymes offer remarkable substrate specificity, biodegradability, and the capacity to degrade a wide array of pollutants, positioning them as powerful tools for bioremediation. However, practical applications are often hindered by limitations in enzyme stability and reusability. Enzyme immobilization techniques have emerged as transformative strategies, enhancing enzyme stability and reusability by anchoring them onto inert or activated supports. These improvements lead to more efficient pollutant degradation and cost-effective bioremediation processes. This review delves into the diverse immobilization methods, showcasing their success in degrading various environmental pollutants, including pharmaceuticals, dyes, pesticides, microplastics, and industrial chemicals. By highlighting the transformative potential of microbial immobilized enzyme biocatalysts, this review underscores their significance in achieving a cleaner and more sustainable future through the mitigation of micropollutant contamination. Additionally, future research directions in areas such as enzyme engineering and machine learning hold immense promise for further broadening the capabilities and optimizing the applications of immobilized enzymes in environmental cleanup. [ABSTRACT FROM AUTHOR]

Objective: Accurate decisions regarding the asthma control level are critical in asthma management. However, an objective laboratory parameter has not yet been defined for detecting asthma control levels in children. Materials and Methods: We aimed to determine objective laboratory parameters that can be used in evaluating the asthma control level. To achieve this, we compared the Global Initiative for Asthma (GINA)-defined asthma control scale with the Pediatric Asthma Control Test and laboratory parameters including serum periostin, tryptase, urinary leukotriene E4, and fractional exhaled nitric oxide levels in determining the control level of asthma in 160 children with asthma. Results: The serum periostin level and FeNO level were significantly high and the median Pediatric Asthma Control Test score was significantly low in uncontrolled patients (p<0.001, p=0.003, p<0.001, respectively). After ROC analysis, p-ACT (AUC:0.914, %95CI:0.86-0.97, p<0.001), serum periostin (AUC:0,669, %95CI:0.59-0.75, p=0.001) and FeNO (AUC:0.755, %95CI:0.67-0.84, p<0.001) were found to be predictive in the assessment of asthma control. There was inconsistency between the GINA-defined asthma control scale and the Pediatric Asthma Control Test in 28.7% of the study group. Within the patients having controlled asthma according to both the GINA-defined asthma control scale and Pediatric Asthma Control Test, 8.7% had high levels of periostin and FeNO. Besides, serum periostin levels and FeNO levels were both normal in 25.0% of the patients with uncontrolled asthma according to the GINAdefined asthma control scale and the Pediatric Asthma Control Test. Conclusion: The asthma control status demonstrated a correlation with FeNO and serum periostin levels. We hold the belief that incorporating objective laboratory parameters, such as FeNO and serum periostin, for the assessment of asthma control levels may have the potential to mitigate both overtreatment and undertreatment in the management of asthma. [ABSTRACT FROM AUTHOR]

The bacteria producing proteases are important for the industries. The proteases are utilized commercially in leather, detergent, pharmaceutical and food industry. The proteases are hydrolytic enzymes which can degrade protein into peptides and amino acids. In leather industry proteases are employed to remove the redundant parts from the animal hide especially hairs to make fine quality of the products. The proteases also prove as green environmental approach. In present study a total of 40 bacterial isolates were recovered from the soil samples of the tannery and screened for proteolytic activity on casein ager plates, among them three isolates were selected with good activity. The morphological and biochemical characteristic features were used to identify the strains; the different conditions of the culture and medium were optimized to check the protease activity. The best proteolytic activity was observed at temperature of 37 °C, pH 8.5, 6% casein concentration as substrate and casein as nitrogen sources as well at 12 h of incubation. The maximum activities by SZ2, SZ1 and SZ3 observed 16.53U/mL, 8U/mL and 7.16U/mL, respectively. The goat skin was treated with proteases from these isolates, complete dehairing observed after 12 h of incubations. Present study was conducted to identify microbes from local tannery, to find out most efficient strain for protease production and to use these enzymes in leather industry. [ABSTRACT FROM AUTHOR]

Trichoderma reesei RUT-C30 was cultivated on differentially pretreated rice straw and pure cellulose as a carbon source/inducer for cellulase production, and the enzymes were evaluated for hydrolysis of sequential acid and alkali pretreated rice straw. Growth on pretreated rice straw enhanced protein secretion and cellulase activities compared to pure cellulose as a carbon source. The yield of cellulolytic enzymes was higher for alkali pretreated rice straw (ALP-RS), while H2O2-treated (HP-RS) could not induce cellulases to a larger level compared to pure cellulose. Protein concentration was 3.5-fold higher on ALP-RS as compared to pure cellulose, with a maximum filter-paper cellulase (FPase) activity of 1.76 IU/ml and carboxy-methyl cellulase (CMCase) activity of 40.16 IU/ml (2.18 fold higher). Beta-glucosidase (BGL) activity was more or less the same with the different substrates and supplementation of heterologous BGL could result in a quantum jump in hydrolytic efficiencies, which in the case of ALP-RS induced enzymes was 34% (increased from 69.26% to 92.51%). The use of lignocellulosic biomass (LCB) itself as a substrate for the production of cellulase is advantageous not only in terms of raw material costs but also for obtaining a more suitable enzyme profile for biomass hydrolysis. [ABSTRACT FROM AUTHOR]

APOBEC proteins are cytidine deaminases that restrict the replication of viruses and transposable elements. Several members of the APOBEC3 family, APOBEC3A, APOBEC3B, and APOBEC3H-I, can access the nucleus and cause what is thought to be indiscriminate deamination of the genome, resulting in mutagenesis and genome instability. Although APOBEC3C is also present in the nucleus, the full scope of its deamination target preferences is unknown. By expressing human APOBEC3C in a yeast model system, I have defined the APOBEC3C mutation signature, as well as the preferred genome features of APOBEC3C targets. The APOBEC3C mutation signature is distinct from those of the known cancer genome mutators APOBEC3A and APOBEC3B. APOBEC3C produces DNA strand-coordinated mutation clusters, and APOBEC3C mutations are enriched near the transcription start sites of active genes. Surprisingly, APOBEC3C lacks the bias for the lagging strand of DNA replication that is seen for APOBEC3A and APOBEC3B. The unique preferences of APOBEC3C constitute a mutation profile that will be useful in defining sites of APOBEC3C mutagenesis in human genomes. [ABSTRACT FROM AUTHOR]

Deficiency of Adenosine Deaminase 2 (DADA2) is a systemic disorder that is inherited in an autosomal recessive manner. Initially identified as a monogenic form of systemic vasculitis that mirrors polyarteritis nodosa (PAN), DADA2 is attributed to biallelic pathogenic variants in the adenosine deaminase 2 gene (ADA2; formerly known as CECR1) on chromosome 22q11. DADA2 presents with variable symptoms that can manifest as vasculitis, immune dysfunction, or hematological abnormalities. The primary disease phenotypes consist of polyarteritis nodosa (PAN)-like vasculitis, Diamond-Blackfan anemia (DBA)-like hematologic findings, and immunodeficiency. The vasculitic condition often manifests through severe ischemic or hemorrhagic strokes that can be life-threatening. To confirm the diagnosis of DADA2, it is imperative to analyze the ADA2 gene and measure low or unnoticeable ADA2 activity in plasma or serum. The use of anti-tumor necrosis factor agents has proven to be efficacious in treating patients with an inflammatory phenotype. Hematopoietic stem cell transplantation (HSCT) stands out as a promising treatment alternative for patients presenting with predominant hematologic or immunologic manifestations. [ABSTRACT FROM AUTHOR]