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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.

Carboxylesterases comprise a major class of α/β‐fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/β‐hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C‐terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/β‐hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C‐terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C‐terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase‐like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases. [ABSTRACT FROM AUTHOR]

In recent years, the use of fish-derived probiotics in aquaculture has become more widespread. However, research on Anguilla japonica -derived probiotics is still limited. To evaluate the potential of probiotics for disease control in eel aquaculture, isolates were obtained from the intestinal tract of healthy Anguilla japonica. These isolates were assessed for their adhesion properties, inhibition of pathogen adhesion, and hydrolytic enzyme production. Morphological characteristics and 16S rRNA sequence analysis were used for identification. Results showed that the AJQ03 strain adhered to the intestinal mucus and inhibited common pathogenic bacteria through adhesion inhibition, and further produced amylase, lipase, protease, and cellulase. Based on morphological characteristics and 16S rRNA sequencing, AJQ03 was identified as Bacillus subtilis. The strain demonstrated tolerance to various extreme conditions, as well as survival in simulated gastrointestinal fluids and superior growth in intestinal fluid compared to Luria-Bertani (LB) broth. In vitro safety tests showed that AJQ03 was not resistant to 32 antibiotics and exhibited γ hemolysis on blood plate. In vivo safety tests demonstrated a 100% survival rate for the fish, with stable organ indices, reduced bacterial loads in the liver and spleen, and complete bacterial clearance by day 7 without residue. Intestinal bacterial load results confirmed effective colonization by strain AJQ03. Analysis of the impact of AJQ03 on the gut microbiota of A. japonica revealed a significant increase in the relative abundance of Bacillus at the genus level, corroborating the colonization efficiency of AJQ03. Additionally, the relative abundances of Klebsiella , Pseudomonas , and Aeromonas were significantly lower compared to the controls, indicating that strain AJQ03 effectively reduced harmful bacteria and improved gut microbiota composition. This study confirms that B. subtilis AJQ03, isolated from the intestine of A. japonica , can serve as a probiotic candidate in A. japonica aquaculture. [ABSTRACT FROM AUTHOR]

Saline shallow lakes in arid and semi-arid regions frequently undergo drying episodes, leading to significant variations in salinity and water availability. Research on the impacts of salinity and drought on the structure and function of biofilms in hypersaline shallow lakes is limited. This study aimed to understand the potential changes of biofilms in playa-lake sediments during the drying process. Sediments were sampled at different depths (surface, subsurface) and hydrological periods (wet, retraction, and dry), which included a decrease in water activity (aw, the availability of water for microbial use) from 0.99 to 0.72. aw reduction caused a greater effect on functional variables compared to structural variables, indicating the high resistance of the studied biofilms to changes in salinity and water availability. Respiration and hydrolytic extracellular enzyme activities exhibited higher values under high aw, while phenol oxidase activity and prokaryote biomass increased at lower aw. This shift occurred at both depths but was more pronounced at the surface, possibly due to the more extreme conditions (up to 0.7 aw). The increased levels of extracellular polymeric substances and carotenoids developed at low aw may help protect microorganisms in high salinity and drought environments. However, these harsh conditions may interfere with the activity of hydrolytic enzymes and their producers, while promoting the growth of resistant prokaryotes and their capacity to obtain C and N sources from recalcitrant compounds. The resilience of biofilms in hypersaline lakes under extreme conditions is given by their resistant biochemichal structure and the adaptability of their microbial functioning. [ABSTRACT FROM AUTHOR]

During challenge of Caenorhabditis elegans with human bacterial pathogens such as Pseudomonas aeruginosa and Enterococcus faecalis , the elicited host response can be damaging if not properly controlled. The activation of Nrf (nuclear factor erythroid-related factor)/CNC (Cap-n-collar) transcriptional regulators modulates the response by upregulating genes that neutralize damaging molecules and promote repair processes. Activation of the C. elegans Nrf ortholog, SKN-1 , is tightly controlled by a myriad of regulatory mechanisms, but a central feature is an activating phosphorylation accomplished by the p38 mitogen-activated kinase (MAPK) cascade. In this work, loss of CDC-48, an AAA+ ATPase, was observed to severely compromise SKN-1 activation on pathogen and we sought to understand the mechanism. CDC-48 is part of the endoplasmic reticulum (ER)-associated degradation (ERAD) complex where it functions as a remodeling chaperone enabling the translocation of proteins from the ER to the cytoplasm for degradation by the proteosome. Interestingly, one of the proteins retrotranslocated by ERAD, a process necessary for its activation, is SKN-1A, the ER isoform of SKN-1. However, we discovered that SKN-1A is not activated by pathogen exposure in marked contrast to the cytoplasmic-associated isoform SKN-1C. Rather, loss of CDC-48 blocks the antioxidant response normally orchestrated by SKN-1C by strongly inducing the unfolded protein response (UPRER). The data are consistent with the model of these 2 pathways being mutually inhibitory and support the emerging paradigm in the field of coordinated cooperation between different stress responses. [ABSTRACT FROM AUTHOR]

Vasculitis in children represents a diverse group of diseases characterized by inflammation of blood vessels, which can lead to significant morbidity if not promptly recognized and managed. This review explores the epidemiology, pathophysiology, classification, and management of key pediatric vasculitides. Classification based on vessel size aids in diagnosis and treatment. Understanding these conditions” clinical features and therapeutic options is critical for improving pediatric patient outcomes and preventing long-term complications. Continued research is essential for refining treatment strategies and enhancing patient care. [ABSTRACT FROM AUTHOR]

BACKGROUND: Traumatic brain injury (TBI)-related morbidity is caused largely by secondary injury resulting from hypoxia, excessive sympathetic drive, and uncontrolled inflammation. Aeromedical evacuation (AE) is used by the military for transport of wounded soldiers to higher levels of care. We hypothesized that the hypobaric, hypoxic conditions of AE may exacerbate uncontrolled inflammation after TBI that could contribute to more severe TBI-related secondary injury. STUDY DESIGN: Thirty-six female pigs were used to test TBI vs Sham TBI, hypoxia vs normoxia, and hypobaria vs ground conditions. TBI was induced by controlled cortical injury, hypobaric conditions of 12,000 ft were established in an altitude chamber, and hypoxic exposure was titrated to 85% SpO2 while at altitude. Serum cytokines, ubiquitin C-terminal hydrolase L1, and TBI biomarkers were analyzed via ELISA. Gross analysis and staining of cortex and hippocampus tissue was completed for glial fibrillary acidic protein and phosphorylated tau. RESULTS: Serum interleukin-1β, interleukin-6, and tumor necrosis factor-α were significantly elevated after TBI in pigs exposed to altitude-induced hypobaria/hypoxia, as well as hypobaria alone, compared with ground level/normoxia. No difference in TBI biomarkers after TBI or hypobaric, hypoxic exposure was noted. No difference in brain tissue glial fibrillary acidic protein or phosphorylated tau when comparing the most different conditions of Sham TBI + ground or normoxia with the TBI + hypobaria/hypoxia group was noted. CONCLUSIONS: The hypobaric environment of AE induces systemic inflammation after TBI. Severe inflammation may play a role in exacerbating secondary injury associated with TBI and contribute to worse neurocognitive outcomes. Measures should be taken to minimize barometric and oxygenation changes during AE after TBI. [ABSTRACT FROM AUTHOR]

Glutaminases are a unique group of hydrolytic enzymes with potential applications in cancer therapy and the food industry. This study focused on the biological characterisation and computational conformation dynamics of YLaM, a potential L-glutaminase from a halothermotolerant Bacillus licheniformis. The research integrated experimental and computational methods to identify the YLaM sequence and predict its structural and functional properties. YLaM was amplified using a polymerase chain reaction, cloned into a pET-22b(+) vector, sequenced, and submitted to BankIt/NCBI. This experimental work was complemented by in silico analysis, including structural modelling via the I-TASSER web server, molecular docking using AutoDockTools 1.5.6, and molecular dynamic (MD) simulations using GROMACS v5.1.4. The study resulted in the development of a high-quality 3D model of YLaM through homology modelling and structural refinement, enabling a detailed exploration of its binding affinity with L-glutamine (L-Gln). By identifying active site residues using the homotetramer structure of human glutaminase, crucial interactions with L-Gln were validated, confirming its catalytic function. Finally, MD simulations confirmed the stability of the complex under physiological conditions. This combined molecular identification and functional simulations provide comprehensive insights into the physicochemical properties of the enzyme, offering valuable information for subsequent assessments, including recombinant production and biological impact evaluations. [ABSTRACT FROM AUTHOR]

E. coli and total coliforms are the most widely used indicator organisms for microbial monitoring of drinking water and recreational freshwater. In many remote and low-resource settings, however, conventional laboratory methods for quantifying these indicators are challenging or infeasible to perform due to limited access to laboratory facilities. The availability of rapid, low-cost methods for quantifying indicator organisms in freshwater samples without the need for laboratory facilities is crucial to facilitate the rapid and robust monitoring of microbial water quality in these types of settings. The global misuse and abuse of antimicrobials have contributed to the rise of antimicrobial resistance. Thus, simple culture methods are needed to detect indicators of such bacteria in freshwaters. In 2021, the World Health Organization released the Tricycle protocol to address this issue by providing guidance for culture-based detection of extended-spectrum beta-lactamase (ESBL)-producing E. coli in environmental samples. Our research goal was to compare the 100-ml sample volume ESBL E. coli quantal and enumerative commercial tests against the more complex Tricycle protocol to detect and quantify ESBL E. coli in surface waters. Both commercial tests gave results comparable with the results obtained using the Tricycle protocol, and the quantal and enumerative commercial tests were easier and faster to perform than the Tricycle protocol. [ABSTRACT FROM AUTHOR]

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

An intense black pigmented halotolerant yeast GUBPC1, was obtained from the solar salterns of Nerul, Goa-India. The isolate could tolerate 0 to 20 % NaCl. FE-SEM analysis revealed its polymorphic nature, exhibiting oval cells at higher salt concentrations and filamentous spindle like shapes at lower concentrations. Initially, the cells appear oval, yeast like in shape but gradually after 15 days of incubation, it becomes elongated and undergoes budding, exhibiting various budding patterns, from single polar bud to bipolar buds with annellidic ring, to lateral buds and eventually forming filamentous hyphae. The intracellular black pigment was identified as melanin based on ultraviolet–visible spectroscopy analysis. The molecular identification of the culture showed closest similarity with Hortaea werneckii. Plant polymer-degrading enzymatic activities such as cellulase, laccase, chitinase, xylanase, pectinase, amylase and protease were exhibited by the isolate GUBPC1. To further understand and explore its biotechnological potential, we performed whole-genome sequencing and analysis. The obtained genome size was 26.93 Mb with 686 contigs and a GC content of 53.24 %. We identified 9383 protein-coding genes, and their functional annotation revealed the presence of 435 CAZyme genes and 16 functional genes involved in secondary metabolite synthesis, thus providing a basis for its potential value in various biotechnological applications. [ABSTRACT FROM AUTHOR]

In this study, we focused on reducing the molecular weight of purified red okra pectin using various hydrolytic enzymes and evaluating its physicochemical properties or characterization. The enzyme treatments targeted both the main pectin chain and the side-chain sugars, resulting in a reduction in the molecular weight by approximately 10% (from 647 kDa) to 60% (down to 252 kDa). Both the purified red okra pectin and enzyme-treated pectins exhibited a homogalacturonan (HG)-type backbone. Fourier transform infrared (FT-IR) spectroscopy revealed a decrease in the absorbance peak for the pectin backbone (1200–1000 cm−1) in the low-molecular-weight (LMW) pectin. The most significant decrease was observed at 3300 cm−1 in pectin treated with both RGH+RGAE enzymes, indicating reduced sugar bonds. These results demonstrate the physicochemical changes in LMW red okra pectin following enzyme treatment and confirm its potential applications due to its unique characteristics. [ABSTRACT FROM AUTHOR]

Control of fungal phytopathogens is a significant challenge in modern agriculture. The widespread use of chemical fungicides to control these pathogens often leads to environmental and food contamination. An eco-friendly alternative that can help reduce reliance on these chemicals is plant growth–promoting bacteria (PGPB), particularly those of the genus Paenibacillus, which appear to be highly effective. The review aims to summarize the existing knowledge on the potential of Paenibacillus spp. as fungal biocontrol agents, identify knowledge gaps, and answer whether other species of the genus Paenibacillus, in addition to Paenibacillus polymyxa, can also be effective biocontrol agents. Paenibacillus spp. can combat plant phytopathogens through various mechanisms, including the production of lipopeptides (such as fusaricidin, paenimyxin, and pelgipeptin), the induction of systemic resistance (ISR), hydrolytic enzymes (chitinase, cellulase, and glucanase), and volatile organic compounds. These properties enable Paenibacillus strains to suppress the growth of fungi such as Fusarium oxysporum, F. solani, Rhizoctonia solani, Botrytis cinerea, or Colletotrichum gloeosporioides. Notably, several strains of Paenibacillus, including P. polymyxa, P. illinoisensis KJA-424, P. lentimorbus B-30488, and P. elgii JCK1400, have demonstrated efficacy in controlling fungal diseases in plants. Importantly, many formulations with Paenibacillus strains have already been patented, and some are commercially available, but most of them contain only P. polymyxa. Nevertheless, considering the data presented in this review, we believe that other strains from the Paenibacillus genus (besides P. polymyxa) will also be commercialized and used in plant protection in the future. Importantly, there is still limited information regarding their impact on the native microbiota, particularly from the metataxonomic and metagenomic perspectives. Expanding knowledge in this area could enhance the effectiveness of biocontrol agents containing Paenibacillus spp., ensuring safe and sustainable use of biological fungicides. [ABSTRACT FROM AUTHOR]

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts. [ABSTRACT FROM AUTHOR]

Background: Cooling towers (CTs) are crucial to myriad industrial processes, supporting thermal exchange between fluids in heat exchangers using water from lakes and rivers as coolant. However, CT water can sometimes introduce microbial contaminants that adhere to and colonize various surfaces within the CT system. These microorganisms can form biofilms, significantly hindering the system's thermal exchange efficiency. Current treatment strategies employ oxidizing biocides to prevent microbial growth. However, despite their affordability, they do not eliminate biofilms effectively and can lead to corrosive damage within the system. Herein, we aim to devise an anti-biofilm strategy utilizing hydrolytic enzymes (such as α-amylase, glucoamylase, pectin-lyase, cellulase, protease, and DNase) alongside antibiotics (including meropenem, ciprofloxacin, gentamicin, erythromycin, chloramphenicol, and ceftriaxone) to combat microbial growth and biofilm formation in cooling systems. Results: All enzymes reduced the development of the biofilms significantly compared to controls (p < 0.05). The polysaccharidases exhibited biomass reduction of 90%, except for pectin-lyase (80%), followed by DNAse and protease at 43% and 49%, respectively. The antibiotics reduced the biofilms of 70% of isolates in concentration of > 2 mg/mL. The minimal biofilm eradication concentration (MBEC) lower than 1 mg/mL was detected for some 7-day-old sessile isolates. The enzymes and antibiotics were also used in combination against biofilms using the modified Chequerboard method. We found six synergistic combinations, with Fractional inhibitory concentrations (FIC) < 0.5, out of the ten tested. In the presence of the enzymatic mixture, MBECs presented a significant decrease (p < 0.05), at least 4-fold for antibiotics and 32-fold for enzymes. Moreover, we characterized high molecular weight (> 12 kDa) exopolysaccharides (EPS) from biofilms of ten isolates, and glycosyl composition analysis indicated a high frequency of glucose, mannose, erythrose, arabinose, and idose across isolates EPS contrasting with rhamnose, allose, and those carbohydrates, which were detected in only one isolate. Conclusion: The synergistic approach of combining enzymes with antibiotics emerges as a highly effective and innovative strategy for anti-biofilm intervention, highlighting its potential to enhance biofilm management practices. [ABSTRACT FROM AUTHOR]

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]

Parasitic species of Trichoderma use hydrolytic enzymes to destroy the host cell wall. Preferent carbon and nitrogen sources suppress the expression of genes related to parasitism. Here, different nutrients were evaluated in the parasitic isolated NI-1, which was identified as Trichoderma atroviride. The genes cbh1 and chb2 (cellobiohydrolases), bgl3.1 (endoglucanase), and pra1 and prb1 (proteinases) were poorly expressed during the interaction between NI-1 and Phytophthora capsici on PDA. However, gene expression improved on minimal medium with preferent and alternative carbon sources. Dextrin and glucose stimulated higher transcript levels than cellulose, sucrose, and glycerol. Also, ammonium stimulated a stronger parasitic responsiveness than the alternative nitrogen sources. During interaction against different phytopathogens, NI-1 detects their host differentially from a distance due to the cbh1 and cbh2 genes being only induced by P. capsici. The pra1 and ech42 genes were induced before contact with Botrytis cinerea and Rhizoctonia solani, while when confronted with P. capsici they were stimulated until contact and overgrowth. The prb1 and bgl3.1 genes were induced before contact against the three-host assayed. Overall, T. atroviride prefers to parasitize and has the capacity to distinguish between an oomycete and a fungus, but nutrient quality regulates its parasitic responsiveness. [ABSTRACT FROM AUTHOR]

This review covers, for the first time, all methods based on the use of Aspergillus strains as biocontrol agents for the management of plant diseases caused by fungi and oomycetes. Atoxigenic Aspergillus strains have been screened in a variety of hosts, such as peanuts, maize kernels, and legumes, during the preharvest and postharvest stages. These strains have been screened against a wide range of pathogens, such as Fusarium, Phytophthora, and Pythium species, suggesting a broad applicability spectrum. The highest efficacies were generally observed when using non-toxigenic Aspergillus strains for the management of mycotoxin-producing Aspergillus strains. The modes of action included the synthesis of antifungal metabolites, such as kojic acid and volatile organic compounds (VOCs), secretion of hydrolytic enzymes, competition for space and nutrients, and induction of disease resistance. Aspergillus strains degraded Sclerotinia sclerotiorum sclerotia, showing high control efficacy against this pathogen. Collectively, although two Aspergillus strains have been commercialized for aflatoxin degradation, a new application of Aspergillus strains is emerging and needs to be optimized. [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]

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]

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]

Background: Recent studies have described higher incidences of concussion, with more severe symptoms and worse outcomes in female athletes compared with male athletes. Purpose: To compile current knowledge about sex-specific differences in incidence, biomechanics, biomarkers, imaging, and outcomes of concussion in athletes participating in contact sports to better understand which fields should be explored in more detail. Study Design: Scoping review; Level of evidence, 3. Methods: The PubMed database was searched for articles published between January 2000 and November 2020 using the Medical Subject Headings terms "craniocerebral trauma" and "brain concussion" combined with the contact sports "football,""soccer,""hockey," and "boxing." Eligibility criteria were based on the recommendations of the Scottish Intercollegiate Guidelines Network. It focused on sex-specific differences within 5 major topics: (1) epidemiology, (2) biomechanics, (3) biomarkers, (4) imaging, and (5) specific concussion outcome variables, including neurocognitive performance, injury severity, and behavioral and psychological symptoms. Results: A total of 22 studies were included. Eight studies investigated the incidence of concussion, with 4 of the 8 finding a significantly higher incidence rate for female versus male athletes. Six studies that focused on biomechanics found that female athletes received fewer impacts with lower magnitudes. One study addressed biomarkers, showing that S100 calcium-binding protein B and neuron-specific enolase were increased after a game in female athletes, and the level of increase was similar to the changes found in male athletes. Based on the 3 imaging studies, affected brain tissue was greatest in areas associated with tau pathology in chronic traumatic encephalopathy. One study showed a lower hypointensity burden index after a season of ice hockey for female athletes, while another study showed more regions with white matter alterations. Seven studies examined concussion outcomes, with 4 studies showing more severe neuropsychological deficits; in addition, female athletes reported more and worse symptoms than male athletes. Conclusion: The results of this review indicated that female athletes had a higher risk of sustaining a concussion, although they received fewer impacts with lower magnitudes than male athletes. Biomarkers were able to be used equally for both sexes. Female athletes also had a higher neuropsychological deficit and increasingly worse symptoms after a concussion. [ABSTRACT FROM AUTHOR]

Lipases are hydrolytic enzymes which have significant potential for commercial applications, particularly in the breakdown of oil contaminants. Serratia marcescens EGHK-19 isolate exhibited considerable lipase activity. This study investigates the optimization, purification, and characterization of lipase from the Serratia marcescens EGHK-19 isolate. The optimized culture conditions revealed that maximal lipase activity was achieved after 24 hours at 30°C and pH 7, with continuous shaking at 150 rpm. Utilizing a 2% inoculum percentage with 1% diesel and 0.3% tryptone in the presence of Fe2+, Ca2+, Mg2+ salts, and Tween 80 resulted in the highest activity at 17.278 U/ml/min. The purification process involved acetone precipitation and DEAE-Sephadex column chromatography, revealing a molecular weight of approximately 60 kDa on SDS-PAGE. This method exhibited a 0.985-fold purification, and the final yield was limited to 2.097% due to lipase aggregates. Characterization of the purified lipase indicated optimal activity (8.765 U/mL/min) at 40°C a nd p H 7. T he K m a nd V max values w ere c alculated a s 6 .89 m M a nd 6 5.79 µmol/min, respectively. The presence of SDS, Tween 80, and Triton X-100 surfactants resulted in the inhibition of lipase activity. Despite these insuggestss, the biochemical characteristics of the purified lipase suggest its potential as an excellent candidate for various industrial applications. [ABSTRACT FROM AUTHOR]

Candida species are opportunistic fungal pathogens commonly found in clinical settings, causing a spectrum of infections known as candidiasis. The virulence of these organisms is multifunctional, involving attributes such as adhesion, biofilm formation, secretion of hydrolytic enzymes and evasion of host immune responses. Evaluating the Candida albicans and non-albicans Candida species isolated in different clinical samples and their in vitro antifungal susceptibity profile. This was a 1 years prospective observational study conducted in the Department of Microbiology at Integral Institute of Medical Sciences & Research, Lucknow. The sample size of this study was 1130. Culture identification, specification, antifungal susceptibility testing was conducted in Microbiology Department according to the CLSI guidelines. Out of 1130 different samples, 67.5% (652) were culture positive, among them only 21.1% (138) were Candida isolates. Out of which 53(38.4%) were Candida albicans, while 85(61.6%) were Non-Candida albicans. Among Non-Candida albicans, the frequency of Candida tropicalis was 58 (68.2%). Maximum number of Non-Candida albicans were isolated from Urine samples (44.7%) followed by Vaginal swab(22.3%) and sputum(20%) While frequency of C.albicans was found more in Sputum sample (39.6%), followed by urine (24.5%), Vaginal swab (22.6%). It was observed that 112(53.5%) Candida isolates shows biofilm production, While Phospholipidase enzymes production were observed in only 21(10.04%) of Candida isolates. It was observed that the maximum sensitivity was observed in Amphotericin-B(95%), followed by Voriconazole(85%) and itraconazole(49.2%). In conclusion, characterization of virulence factors in Candida species isolated from various clinical specimens provides valuable insights into the pathogenesis and clinical outcomes of candidiasis. Continued research efforts are essential to address the evolving challenges posed by Candida infections and improve patient outcomes in clinical practice. [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]

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]

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]

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]

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]