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Pigeonpea ( Cajanus cajan ) urease was immobilized on 1 cm×1 cm DEAE-cellulose paper strips. The optimum immobilization (51% activity) was observed at 4 °C, with a protein concentration of 1.0 mg/strip. The apparent optimum pH shifted from 7.3 to 6.8. Immobilized urease showed an optimal stability temperature of 67 °C, compared with 47 °C for the soluble urease. Time-dependent kinetics of the thermal inactivation of the immobilized urease were examined and found to be monophasic as compared with the soluble enzyme, which was biphasic. The Michaelis constant ( K m) for the DEAE-cellulose-immobilized urease was found to be 4.75 mM, 1.5 times higher than the soluble enzyme. Immobilized strips stored at 4 °C showed an increased half-life ( t 1/2=150 days). There was practically no leaching of the enzyme from the immobilized strips over a period of 2 weeks. These strips were used for estimating the urea content of blood samples; the results obtained matched well with those obtained in a clinical laboratory through an Autoanalyzer® (Zydus Co., Rome, Italy). The easy availability of pigeonpea urease, the ease of its immobilization on DEAE-cellulose strips and the significantly lower cost of urease described in the present study makes it a suitable product for future applications in diagnostics. [ABSTRACT FROM AUTHOR]

The delayed consequences of the influence of hepatic encephalopathy (HE) on the metabolism of animals have not been studied enough. We have previously shown that the development of acute HE under the influence of the thioacetamide (TAA) toxin is accompanied by pathological changes in the liver, an imbalance in CoA and acetyl CoA, as well as a number of metabolites of the TCA cycle. This paper discusses the change in the balance of amino acids (AAs) and related metabolites, as well as the activity of glutamine transaminase (GTK) and ω-amidase enzymes in the vital organs of animals 6 days after a single exposure to TAA. The balance of the main AAs in blood plasma, liver, kidney, and brain samples of control (n = 3) and TAA-induced groups (n = 13) of rats that received the toxin at doses of 200, 400, and 600 mg/kg was considered. Despite the apparent physiological recovery of the rats at the time of sampling, a residual imbalance in AA and associated enzymes persisted. The data obtained give an idea of the metabolic trends in the body of rats after their physiological recovery from TAA exposure and may be useful for prognostic purposes when choosing the necessary therapeutic agents. [ABSTRACT FROM AUTHOR]

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation. Author summary: Peptidoglycan biosynthesis is a major target for antibacterials. The covalently closed peptidoglycan mesh, called sacculus, protects the bacterium from lysis due to its turgor. Sacculus growth is facilitated by the balanced activities of synthases and hydrolases, and disturbing this balance leads to cell lysis and bacterial death. Because of the large number and possible redundant functions of peptidoglycan hydrolases, it has been difficult to decipher their individual functions. In this paper we show that the DD-endopeptidase PBP4 localizes at midcell during septal peptidoglycan synthesis in Escherichia coli and is involved in the timing of the assembly and activation of the division machinery. This shows that inhibition of certain hydrolases could weaken the cells and might enhance antibiotic action. [ABSTRACT FROM AUTHOR]

Abstract: The increasing industrial utilization of polyacrylamide to assist water clarification, sludge conditioning, papermaking, and secondary oil recovery leads to environmental pollution. In this work, an acrylamide degrading bacterium was isolated from paper mill effluent at Charan mahadevi, Tamilnadu, India. The minimal medium containing acrylamide (40 mM) served as a sole source of carbon and nitrogen for acrylamide degrading bacteria. The bacterial strain has grown well in 40 mM acrylamide at pH (6–7) at 30 °C. Within 24–48 h acrylamide was converted into acrylic acid and other metabolites. Based on biochemical characteristics and 16S rRNA gene sequence, the bacterial strain was identified as Gram negative, diplobacilli Moraxella osloensis MSU11. The acrylamide hydrolyzing bacterial enzyme acrylamidase was purified by HPLC. The enzyme molecular weight was determined to be approximately 38 kDa by SDS-PAGE using reference enzyme Pectinase. These results show that M. osloensis MSU11 has a potential to degrade the acrylamide present in the environment. [Copyright &y& Elsevier]

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria. The septal peptidoglycan hydrolysis machinery is a crucial structure for bacterial cell division. This study analyses the cryo-EM structure and enzymatic function of E. coli FtsEX, with and without the lytic-factor protein EnvC, characterizing the molecular basis of FtsEX regulation of peptidoglycan hydrolysis, which could be used to develop novel treatments targeting drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. The oncometabolites have been studied in OSCC, but the mechanism of metabolic reprogramming remains unclear. To identify the potential metabolic markers to distinguish malignant oral squamous cell carcinoma (OSCC) tissue from adjacent healthy tissue and study the mechanism of metabolic reprogramming in OSCC. We compared the metabolites between cancerous and paracancerous tissues of OSCC patients by 1HNMR analysis. We established OSCC derived cell lines and analyzed their difference of RNA expression by RNA sequencing. We investigated the metabolism of γ-aminobutyrate in OSCC derived cells by real time PCR and western blotting. Our data revealed that much more γ-aminobutyrate was produced in cancerous tissues of OSCC patients. The investigation based on OSCC derived cells showed that the increase of γ-aminobutyrate was promoted by the synthesis of glutamate beyond the mitochondria. In OSCC cancerous tissue derived cells, the glutamate was catalyzed to glutamine by glutamine synthetase (GLUL), and then the generated glutamine was metabolized to glutamate by glutaminase (GLS). Finally, the glutamate produced by glutamate-glutamine-glutamate cycle was converted to γ-aminobutyrate by glutamate decarboxylase 2 (GAD2). Our study is not only benefit for understanding the pathological mechanisms of OSCC, but also has application prospects for the diagnosis of OSCC. [ABSTRACT FROM AUTHOR]

Background: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. Results: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved –GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. Conclusion: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1. [ABSTRACT FROM AUTHOR]

Global plastic waste accumulation has become omnipresent in public discourse and the focus of scientific research. Ranking as the sixth most produced polymer globally, polyurethanes (PU) significantly contribute to plastic waste and environmental pollution due to the toxicity of their building blocks, such as diisocyanates. In this study, the effects of PU on soil microbial communities over 18 months were monitored revealing that it had marginal effects on microbial diversity. However, Streptomyces sp. PU10, isolated from this PU‐contaminated soil, proved exceptional in the degradation of a soluble polyester‐PU (Impranil) across a range of temperatures with over 96% degradation of 10 g/L in 48 h. Proteins involved in PU degradation and metabolic changes occurring in this strain with Impranil as the sole carbon source were further investigated employing quantitative proteomics. The proposed degradation mechanism implicated the action of three enzymes: a polyester‐degrading esterase, a urethane bond‐degrading amidase and an oxidoreductase. Furthermore, proteome data revealed that PU degradation intermediates were incorporated into Streptomyces sp. PU10 metabolism via the fatty acid degradation pathway and subsequently channelled to polyketide biosynthesis. Most notably, the production of the tri‐pyrrole undecylprodigiosin was confirmed paving the way for establishing PU upcycling strategies to bioactive metabolites using Streptomyces strains. [ABSTRACT FROM AUTHOR]

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km = 43.5 mM−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−1). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales. [ABSTRACT FROM AUTHOR]

Biodegradation of anionic polyacrylamide (HPAM) by key enzymes, such as amidase and laccase, has been reported. However, the interaction details between HPAM and enzymes are still unclear. Here, docking was performed to demonstrate their binding modes and interactions. Then, alanine (ALA) scanning and saturated mutation were performed by virtual mutation based on affinity. Finally, the bioactivities of substrates were evaluated with frontier orbitals. The docking results showed that Rh Amidase-HPAM-2 was the most stable, mainly due to more hydrophobic interactions and hydrogen bonds, while a strong salt bridge and two hydrogen bonds were found to be the main reason for B. subtilis laccase-HPAM-3 being the most stable. It was confirmed by virtual mutations that PHE146, ILE450, LYS96 and GLY193 were the key amino acid residues of Rh Amidase degrading HPAM-2, and the mutant D191A could strengthen their affinity. It was also confirmed that TYR118, TYR133, ARG487 and LYS135 were the key amino acid residues of B. subtilis laccase degrading HPAM-3. The frontier orbital analysis indicated that the bioactivity of HPAM was higher than that of PAA, and the macromolecular chain was difficult to be degraded. A potential mechanism for HPAM degradation was proposed. The amide side chain of HPAM was hydrolyzed by Rh Amidase into carboxyl, and NH4+ was used as a nitrogen source, while the carbon skeleton of HPAM was broken by B. subtilis laccase as a carbon source for microbial growth. These findings provide a theoretical reference for designing highly efficient enzymes. [ABSTRACT FROM AUTHOR]

This study introduces a new type of ene/yne‐reductase from Cyclocybe aegerita with a broad substrate scope including aliphatic and aromatic alkenes/alkynes from which aliphatic C8‐alkenones, C8‐alkenals and aromatic nitroalkenes were the preferred substrates. By comparing alkenes and alkynes, a ∼2‐fold lower conversion towards alkynes was observed. Furthermore, it could be shown that the alkyne reduction proceeds via a slow reduction of the alkyne to the alkene followed by a rapid reduction to the corresponding alkane. An accumulation of the alkene was not observed. Moreover, a regioselective reduction of the double bond in α,β‐position of α,β,γ,δ‐unsaturated alkenals took place. This as well as the first biocatalytic reduction of different aliphatic and aromatic alkynes to alkanes underlines the novelty of this biocatalyst. Thus with this study on the new ene‐reductase CaeEnR1, a promising substrate scope is disclosed that describes conceivably a broad occurrence of such reactions within the chemical landscape. [ABSTRACT FROM AUTHOR]

A type of grafted acrylate copolymer resins, containing 3-oxo-Nallyl-1,2-benzisothiazole-2(3H)-carboxamide monomer and heterocyclic monomers, was synthesized through the copolymerization of methyl methacrylate (MMA) and butyl acrylate (BA) with functional monomers. The structures of the monomers and copolymers were validated by infrared (IR) and 1 H nuclear magnetic resonance (NMR) spectroscopies. The inhibitory activities of the copolymers on algae, bacteria, and barnacle larvae were measured, and the antifouling potencies against marine macrofouling organisms were investigated. The results showed that the grafted resin had significant inhibitory effects on the growth of three marine algae (Isochrysis galbana, Nannochloropsisoculata, and Chlorella pyrenoidosa), and three bacteria (Vibrio coralliilyticus, Staphylococcus aureus,and Vibrio parahaemolyticus). The target copolymers also showed excellent inhibition of the survival of barnacle larvae. Additionally, the release rate of the antifoulant and the results of the marine field tests indicated that the grafted copolymers had outstanding antifouling potency against the attachment of marine macrofouling organisms. [ABSTRACT FROM AUTHOR]

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus, catalyze hydrolysis of β-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-like bond of the four membered β-lactam ring of nitrocefin, an antibiotic β-lactam surrogate. Examination of the catalytic activities of several PSMα3 variants allowed mapping of the active sites on the amyloid fibrils' surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine arrays. Molecular dynamics simulations further illuminate the structural features of β-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed β-lactams by PSMα3 fibrils, and illustrating antibiotic degradation in actual S. aureus biofilms and live bacteria environments. Overall, this study unveils functional amyloids as catalytic agents inducing degradation of β-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms. A number of mechanisms are known to mediate bacterial antibiotic resistance. Here, Arad et al show that amyloid fibrils produced by Staphylococcus aureus rapidly degrade common antibiotic molecules. [ABSTRACT FROM AUTHOR]

In this paper, poly (2-hydroxyethyl methacrylate) (pHEMA) and poly (2-hydroxyethyl methacrylate)-starch (pHEMA-S) composites were prepared from 2-hydroxyethyl methacrylate and different ratio starch. The pHEMA-S showed high thermal stability and biodegradability. The biodegradability of pHEMA-S composites was in the range of 14-22 % for 4 weeks depending on the starch ratio of pHEMA-S. Then, L-ASNase was immobilized onto pHEMA-S composites. The immobilized L-ASNase was investigated with optimum temperature, pH and kinetic parameters. Immobilization improved the pH stability and thermal stability of the enzyme. The K values were 1.58 and 0.56 mM for free and immobilized enzyme, respectively. [ABSTRACT FROM AUTHOR]

L-asparaginase is an enzyme that is mainly used in the medical industry to treat various cancers and in the food industry to reduce acrylamide in starch-containing foods. In order to be used successfully, L-asparaginase must have a certain concentration and a certain purity to give the best results and minimize side effects in the medical industry. The aim of this paper was the screening of different strains of Bacillus, Streptomyces, yeasts and bacterial endophytes for L-asparaginase biosynthesis, and to highlight the methods used to determine the level of L-asparaginase. Several bacterial strains and two yeast strains were selected and the level of L-asparaginase was quantified. At least two microbial strains will be used in further experiments to optimize the enzyme production. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs), which are structurally conserved innate immune molecules in invertebrate and vertebrate animals, play the important roles in regulation of innate immune responses. In this paper, three PGRP genes of spotted sea bass, Lateolabrax maculatus , were cloned, designated as Ssb-PGRP2 , Ssb - PGRP-L2 and Ssb-PGRP-SC2 , respectively. Sequence analysis showed that the deduced amino acid sequences of Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2 proteins contained respectively 468, 482 and 167 amino acid residues, and had the typical structural features of PGRPs, i.e. conserved PGRP domain and Zn2+ binding domain including four specific amino acid residues which were required for amidase activity. q-PCR analysis of total mRNA showed that the mRNA expression of three PGRP genes were detected in all the examined tissues and the expression patterns of Ssb-PGRP2 , Ssb-PGRP-L2 and Ssb-PGRP-SC2 were different. After injected with LPS, Poly (I:C) and Edwardsiella tarda , there was a clear time-dependent expression pattern for each of the three PGRP genes in head kidney, spleen, intestine and gill of the spotted sea bass. In our study, three recombinant proteins corresponding to the three members of the peptidoglycan recognition protein family were expressed and purified. Moreover, all of the three recombinant PGRP proteins significantly inhibited bacterial survival and growth, and expressed bactericidal effects on Vibrio harveyi , Staphylococcus aureus and Edwardsiella tarda. In particular, it was firstly verified that their antimicrobial activity presented the superimposed effect. Overall, these findings indicated that three PGRP genes of spotted sea bass were at least involved in host defense against bacterial infections. • Three members of the PGRP family were identified in spotted sea bass. • The three members were up-regulated by LPS, Poly (I:C) and Edwardsiella tarda. • All of the three recombinant PGRP proteins possessed the amidase activity. • All of the three recombinant proteins expressed bactericidal effects. • Their antimicrobial activity presented the superimposed effect. [ABSTRACT FROM AUTHOR]

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT). [ABSTRACT FROM AUTHOR]

Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Pénicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 A) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH ~6 and 66°C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)- barrel and a smaller ß-sandwich domain. The active site contains an aspartate residue for acid-base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) has a lifetime prevalence of approximately 10% and is one of the most common diseases worldwide. Although many pathogenetic mechanisms of MDD have been proposed, molecular details and a unifying hypothesis of the pathogenesis of MDD remain to be defined. Here, we investigated whether tyrosine nitrosylation, which is caused by reaction of the C-atom 3 of the tyrosine phenol ring with peroxynitrate (ONOO−), plays a role in experimental MDD, because tyrosine nitrosylation may affect many cell functions altered in MDD. To this end, we induced stress through glucocorticoid application or chronic environmental unpredictable stress and determined tyrosine nitrosylation in the hippocampus through immuno-staining and ELISA. The role of catalases and peroxidases for tyrosine nitrosylation was measured using enzyme assays. We show that glucocorticoid- and chronic unpredictable environmental stress induced tyrosine nitrosylation in the hippocampus. Long-term treatment of stressed mice with the classical antidepressants amitriptyline or fluoxetine prevented tyrosine nitrosylation. Tyrosine nitrosylation was also prevented through i.v. application of anti-ceramide antibodies or recombinant ceramidase to neutralize or degrade, respectively, blood plasma ceramide that has been recently shown to induce experimental MDD. Finally, the application of phosphatidic acid, previously shown to be reduced in the hippocampus upon stress, also reverted stress-induced tyrosine nitrosylation. The inhibition of tyrosine nitrosylation by interfering with the formation of NO radicals at least partly restored normal behavior in stressed mice. These data suggest that tyrosine nitrosylation might contribute to the pathogenesis of MDD and targeting this process might contribute to the treatment of MDD. [ABSTRACT FROM AUTHOR]

Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis. GPI-T adds GPI to proteins faithfully despite the sequence variance. Here, the authors reveal structural features underpinning this broad specificity, and a fidelity mechanism where unlocking autoinhibition necessitates the synergistic binding of substrate regions with individually weak specificity. [ABSTRACT FROM AUTHOR]

This paper analyzes the effect of exogenous urea in increased concentration gradient (0, 100, 500 and 1,000 mg L) on photosynthetic pigments (measured spectrophotometrically), uptake of CO (using radioisotope), and urease activity (by measuring ammonia with Nessler's reagent) in leaves of Elodea densa Planch. We have observed that low concentration of urea (100 mg L) stimulates the accumulation of photosynthetic pigments and intensifies photosynthesis in E. densa, whereas high concentration (1,000 mg L) suppresses these processes. Urease activity increased by approximately 2.7 and 8 fold when exogenous urea concentrations were 100 and 500 mg L, respectively. However, exogenous urea in high concentration (1,000 mg L) decreased urease activity by 1.5 fold compared to the control. The necessity of mitigating urea and other nitrogen-containing compounds (NH from urea) in water bodies has been discussed with emphasis on the potential for phytoremediation of urea using common water weed viz. E. densa. [ABSTRACT FROM AUTHOR]

The recently described T6SS (type VI secretion system) acts as a needle that punctures the membrane of the target cells to deliver effector proteins. Type VI amidase effectors can be classified into four divergent families (Tae1-Tae4). These effectors are secreted into the periplasmic space of neighbouring cells via the T6SS and subsequently rupture peptidoglycan. However, the donor cells are protected from damage because of the presence of their cognate immunity proteins [Tai1 (type VI amidase immunity 1)- Tai4]. In the present paper, we describe the structure of Tae3 in complex with Tai3. The Tae3-Tai3 complex exists as a stable heterohexamer, which is composed of two Tae3 molecules and two Tai3 homodimers (Tae3-Tai34-Tae3). Tae3 shares a common NlpC/P60 fold, which consists of N-terminal and C-terminal subdomains. Structural analysis indicates that two unique loops around the catalytic cleft adopt a closed conformation, resulting in a narrow and extended groove involved in the binding of the substrate. The inhibition of Tae3 is attributed to the insertion of the Ω-loop (loop of α3-α4) of Tai3 into the catalytic groove. Furthermore, a cell viability assay confirmed that a conserved motif (Gln-Asp-Xaa) in Tai3 members may play a key role in the inhibition process. Taken together, the present study has revealed a novel inhibition mechanism and provides insights into the role played by T6SS in interspecific competition. [ABSTRACT FROM AUTHOR]

Tse1 (Tse is type VI secretion exported), an effector protein produced by Pseudomonas aeruginosa, is an amidase that hydrolyses the ? -D-glutamyl-DAP (? -D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6-148)-Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1Å=0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like a +ß fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself. [ABSTRACT FROM AUTHOR]

Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes. [ABSTRACT FROM AUTHOR]

In this paper a device, based on urease-loaded microspheres, is presented. The first task of this work was the optimization of a procedure for the alginate microspheres realization, having a radius as close as possible to the optimal one necessary to achieve the maximum enzyme exploitation. This optimal radius was calculated theoretically through a mathematical model which describes the concentration of substrate (urea) inside the microspheres on the assumption of a diffusion-reaction mechanism. The enzyme-loaded microspheres were successfully tested in a prototypal device aimed at the depletion of urea from a circulating fluid simulating blood flow: the results showed that urea concentration in the circulating fluid drops down to less than 25% of the initial value after 5 h. [ABSTRACT FROM AUTHOR]

The molecular basis of the antitumor selectivity of histone deacetylase inhibitors (HDIs) remains unclear. Centrosomal Aurora-A kinase regulates chromosomal segregation during mitosis. The overexpression or amplification of Aurora-A leads to genetic instability, and its inhibition has shown significant antitumor effects. In this paper, we report that structurally related hydroxamate LAQ824 and SK-7068 induce tumor-selective mitotic defects by depleting Aurora-A. We found that HDI-treated cancer cells, unlike nontransformed cells, exhibit defective mitotic spindles. After HDI, Aurora-A was selectively downregulated in cancer cells, whereas Aurora-B remained unchanged in both cancer and nontransformed cells. LAQ824 or SK-7068 treatment inhibited histone deacetylase (HDAC) 6 present in Aurora-A/heat shock protein (Hsp) 90 complex. Inhibition of HDAC6 acetylated Hsp90 and resulted in dissociation of acetylated Hsp90 from Aurora-A. As a result, Hsp70 binding to Aurora-A was enhanced in cancer cells, leading to proteasomal degradation of Aurora-A. Overall, these provide a novel molecular basis of tumor selectivity of HDI. LAQ824 and SK-7068 might be more effective HDIs in cancer cells with Aurora-A overexpression. [ABSTRACT FROM AUTHOR]

14C-urea breath test (UBT) is considered to be an accurate diagnostic test for the detection of active Helicobacter pylori infection. Various test meals are used in 14C-UBT to slow down gastric emptying, and to enhance the gastric distribution, in order to increase the time and area of contact between microorganisms and the tracer substrate. The aim of the present paper was to evaluate the effect of gastric environment on the performance of 14C-UBT using an alkaline and an acidic liquid test meal having gastric emptying retardant effect. The comparison of 14C-UBT was done with liquid test meals (200 mL water) comprising (i) plain drinking water (PDW); (ii) 1.3 g or 3.0 g citric acid (CA); and (iii) 3.0 g trisodium citrate (TSC). Eighteen patients (37 ± 12 years, range 18–57 years) with complaints of dyspepsia participated in the study. The status of H. pylori was confirmed by histology and rapid urease test. A total of 93 kBq of 14C-urea (0.5 mL) in a gelatin capsule was orally administered along with liquid test meals to the overnight fasting subjects. Breath samples were collected and radioactivity measured. Results were expressed as 14CO2/mmol exhaled CO2 as percentage of administered radioactive urea. Higher acidic gastric environment (pH approx. 2.0) with CA was found to increase the exhaled 14CO2 level in a dose-dependent manner as compared to PDW and TSC meal ( P < 0.05) at all time points. With TSC test meal, the expired 14CO2 level decreased in the lower acidic gastric environment (pH approx. 5.3). The peaks of exhaled 14CO2 with TSC test meal were observed at the same time points as that with PDW and CA test meals. The 14C-UBT with TSC was found to be positive in 77% of patients (10/13). Better interaction between the microbial urease and 14C-urea, caused by a test meal that retards gastric emptying and that changes gastric pH, plays an important role in hydrolysis of the administered 14C-urea by H. pylori urease. © 2005 Blackwell Publishing Asia Pty Ltd [ABSTRACT FROM AUTHOR]

The immobilization of a multi-enzyme extract is an excellent method for multi-step biotransformations. This paper describes how a multi-enzyme extract from Agrobacterium radiobacter , rich in d -hydantoinase and N-carbamyl- d -amino acid amidohydrolase was immobilized on chitin for its application on the synthesis of p-hydroxyphenylglycine. The adsorption derivative showed a higher activity than the covalent one. Compared to the soluble multi-enzymatic extract, the adsorption derivative showed greater pH-stability in the pH range under study. Its optimum pH ranged from 7-8. Furthermore, it showed high activity at low temperature. [ABSTRACT FROM AUTHOR]

The study aimed to purify trypsin from the pyloric caeca of Asian seabass (Lates calcarifer), and investigate its proteolytic capability toward acid-soluble collagen (ASC) in comparison with commercial porcine trypsin (CPT). Trypsin was purified from pyloric caeca, a leftover from the evisceration process, via ammonium sulphate (40–60% saturation) precipitation, and a soybean trypsin inhibitor (SBTI)–Sepharose 4B column. A 18.5-fold purification and a yield of 15.2% were obtained. SDS-PAGE analysis confirmed a single band of trypsin with a molecular weight of 23.5 kDa. Purified trypsin also showed the single band in native-PAGE. The optimal pH and temperature of trypsin for BAPNA (the specific substrate for amidase) hydrolysis were 8.5 and 60 °C, respectively. The trypsin was stable within the pH range of 7.0–9.5 and temperature range of 25–55 °C. Protease inhibition study confirmed that the purified enzyme was trypsin. The purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (kcat) of 0.078 mM and 5.4 s−1, respectively, when BAPNA was used. For the hydrolysis of TAME (the specific substrate for esterase), the Km and Kcat were 0.09 mM and 4.8 s−1, respectively. Partially purified seabass trypsin (PPST) had a slightly lower hydrolysis capacity toward ASC than CPT, as evidenced by the lower degree of hydrolysis and protein degradation when the former was used. Both the α-chain and β-chain became more degraded as the hydrolysis time increased. Based on MALDI-TOP, peptides with MW of 2992-2970 Da were dominant in the hydrolysates. Therefore, seabass trypsin could be used in the production of hydrolyzed collagen. It could have economic importance to the market, by replacing some commercial proteases, which have religious constraints. [ABSTRACT FROM AUTHOR]

The immunosuppressive effects of polyethylene glycol-modified asparaginases from Vibrio succinogenes (PEG-asparaginase VS) and Escherichia coli (PEG-asparaginase EC) have been investigated in mice. Measurements of the mitogen-induced blastogenic responses of splenocytes, harvested 5 days after in vivo administration of the PEG- enzymes, show that PEG-asparaginase VS is not immunosuppressive, whereas PEG- asparaginase EC does cause immunosuppression. Both enzymes cause the spleen to be smaller than the control mice. In mice carrying the L5178Y tumour and its associated LDH-elevating virus, which causes the circulation life of asparaginase VS to be comparable to that of PEG-asparaginase VS, tumour regression and its attendant immunological changes are identical in animals treated with either the native or the modified enzyme. The data presented in this paper, along with independent immunological evidence presented by other workers strongly suggest that PEG-asparaginase VS may be the enzyme of choice for clinical use. [ABSTRACT FROM AUTHOR]

A direct reading, six channel conductivity recording apparatus has been constructed and used to assay enzymes, including some of those found in muscle. This paper gives the essential theory of the method, and indicates that it is a highly versatile assay system. It may be applicable whenever ionic substrates are involved, but this is more easily tested by experiment than by calculation. The manipulation involved in an assay is probably less than in any other method. Conductimetry could be used as a kinetic method for some enzymes (for example amidases), but suffers from restrictions in the amounts of non-reacting ionic species which can be tolerated. Improvements in the temperature control of the apparatus could allow much higher sensitivity to be used. [ABSTRACT FROM AUTHOR]

The peptidoglycan (PG) cell wall produced by the bacterial division machinery is initially shared between the daughters and must be split to promote cell separation and complete division. In gram-negative bacteria, enzymes that cleave PG called amidases play major roles in the separation process. To prevent spurious cell wall cleavage that can lead to cell lysis, amidases like AmiB are autoinhibited by a regulatory helix. Autoinhibition is relieved at the division site by the activator EnvC, which is in turn regulated by the ATP-binding cassette (ABC) transporter-like complex called FtsEX. EnvC is also known to be autoinhibited by a regulatory helix (RH), but how its activity is modulated by FtsEX and the mechanism by which it activates the amidases have remained unclear. Here, we investigated this regulation by determining the structure of Pseudomonas aeruginosa FtsEX alone with or without bound ATP, in complex with EnvC, and in a FtsEX-EnvC-AmiB supercomplex. In combination with biochemical studies, the structures reveal that ATP binding is likely to activate FtsEX-EnvC and promote its association with AmiB. Furthermore, the AmiB activation mechanism is shown to involve a RH rearrangement. In the activated state of the complex, the inhibitory helix of EnvC is released, freeing it to associate with the RH of AmiB, which liberates its active site for PG cleavage. These regulatory helices are found in many EnvC proteins and amidases throughout gram-negative bacteria, suggesting that the activation mechanism is broadly conserved and a potential target for lysis-inducing antibiotics that misregulate the complex. [ABSTRACT FROM AUTHOR]

Background: Engaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues. Methods: Both MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC–MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested. Results: The basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking. Conclusions: In the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment. [ABSTRACT FROM AUTHOR]

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(1) Background: Acetaminophen (APAP), an active component of many analgesic and antipyretic drugs, is one of the most concerning trace contaminants in the environment and is considered as an emergent pollutant of marine and aquatic ecosystems. Despite its biodegradability, APAP has become a recalcitrant compound due to the growth of the global population, the ease of availability, and the inefficient wastewater treatment applied. (2) Methods: In this study, we used a transcriptomic approach to obtain functional and metabolic insights about the metabolization of APAP by a phenol-degrading fungal strain, Penicillium chrysogenum var. halophenolicum. (3) Results: We determined that the transcriptomic profile exhibited by the fungal strain during APAP degradation was very dynamic, being characterized by an abundance of dysregulated transcripts which were proportional to the drug metabolization. Using a systems biology approach, we also inferred the protein functional interaction networks that could be related to APAP degradation. We proposed the involvement of intracellular and extracellular enzymes, such as amidases, cytochrome P450, laccases, and extradiol-dioxygenases, among others. (4) Conclusions: Our data suggested that the fungus could metabolize APAP via a complex metabolic pathway, generating nontoxic metabolites, which demonstrated its potential in the bioremediation of this drug. [ABSTRACT FROM AUTHOR]

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an “inverse” acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (K i = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors. [ABSTRACT FROM AUTHOR]

Objective. To predict and determine the mechanism through which Tiao-Bu-Fei-Shen (TBFS) formula improves glucocorticoid resistance in chronic obstructive pulmonary disease (COPD), using network pharmacology, molecular docking technology, and in vitro studies. Methods. The main active components and associated targets of TBFS were screened using the systems pharmacology database of traditional Chinese medicine database (TCMSP). The main COPD targets were retrieved from the Human Gene (GeneCards) and DrugBank databases. A protein-protein interaction (PPI) network was constructed using the protein interaction platform STRING and Cytoscape 3.6.1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genome Pathway (KEGG) analyses were performed using the biological information annotation database Metascape. Molecular docking was performed using the AutoDock Vina software. THP-1 monocytes were treated with TBFS-containing serum and cigarette smoke extract (CSE) for 48 h, and cell proliferation in each group was determined using cell counting kit-8 (CCK-8). A COPD cell model was constructed by stimulating THP-1 monocytes with CSE for 12 h. A lentivirus vector for RNA interference of histone deacetylase 2 (HDAC2) gene was constructed and transfected into the THP-1 monocytes, and the transfection efficiency was verified using quantitative polymerase chain reaction (qPCR) and western blotting (WB). The expression of HDAC2 in each group of cells was detected using qPCR, and the expression of HDAC2, phosphoinositide-3 kinase (PI3K) p85α, glucocorticoid receptor α (GRα), and P-AKT1 in each group of cells was detected through WB. Results. A total of 344 TBFS active components, 249 related drug targets, 1,171 COPD target proteins, and 138 drug and disease intersection targets were obtained. Visual analysis of the PPI network map revealed that the core COPD targets of TBFS were AKT1, IL-6, TNF, TP53, and IL1-β. KEGG pathway enrichment analysis resulted in the identification of 20 signaling pathways as the main pathways involved in the action of TBFS against COPD, including the PI3K-Akt, TNF, and IL-17 signaling pathways. Molecular docking experiments revealed a strong binding capacity of kaempferol, luteolin, and quercetin to the ATK1 protein in TBFS, with quercetin performing the best. PCR results showed that treatment with TBFS significantly increased the expression levels of HDAC2 in the COPD model. WB results showed that TBFS treatment significantly increased the expression levels of GRα and HDAC2 in the COPD model, while reducing the expression levels of P-AKT1. Conclusion. TBFS treatment improves glucocorticoid resistance observed in COPD through downregulation of the PI3K-Akt signaling pathway and promotion of GRα expression. [ABSTRACT FROM AUTHOR]

Pancreatic and lung cancers frequently develop resistance to chemotherapy-induced cell apoptosis during the treatment, indicating that targeting nonapoptotic-related pathways, such as pyroptosis, can be an alternative cancer treatment strategy. Pyroptosis is a gasdermin-driven lytic programmed cell death triggered by inflammatory caspases when initiated by canonical or noncanonical pathways that has been recently seen as a potential therapeutic target in cancer treatment. However, overcoming chemoresistance in cancers by modulating pyroptosis has not been explored. Here, we demonstrate that β5-integrin represses chemotherapy-induced canonical pyroptosis to confer cancer chemoresistance through ASAH2-driven sphingolipid metabolic reprogramming. Clinically, high β5-integrin expression associates with poor patient prognosis and chemotherapeutic responses in cancers. In addition, chemoresistant cells in vitro fail to undergo chemotherapy-induced pyroptosis, which is controlled by β5-integrin. Mechanistically, proteomic and lipidomic analyses indicate that β5-integrin up-regulates sphingolipid metabolic enzyme ceramidase (ASAH2) expression through Src–signal transducer and activator of transcription 3 (STAT3) signaling, which then reduces the metabolite ceramide concentration and subsequent ROS production to prohibit chemotherapy-induced canonical pyroptosis. Using cancer cell lines, patient-derived tumor organoids, and orthotopic lung and pancreatic animal models, we show that administration of a Src or ceramidase inhibitor rescues the response of chemoresistant pancreatic and lung cancer cells to chemotherapy by reactivating pyroptosis in vitro and in vivo. Overall, our results suggest that pyroptosis-based therapy is a means to improve cancer treatment and warrants further investigation. Putting back pyroptosis: While chemotherapeutic drugs are the standard of care for several cancers, patients often develop resistance to apoptotic directed cell death. Pyroptosis, a gasdermin dependent programmed necrotic death, offers a potential target to overcome this chemoresistance. Su et al. have determined that beta 5-integrin represses chemotherapy-induced canonical pyroptosis to induce chemoresistance mediated by sphingolipid metabolic reprogramming. This pathway upregulates the sphingolipid metabolic enzyme ceramidase (ASAH2) that when inhibited in vivo was able to resensitize xenografts to chemotherapy. These results suggest pyroptosis-based therapy as a potential treatment to overcome chemotherapy resistance and warrants further investigation. –DH [ABSTRACT FROM AUTHOR]

Introduction. Acute pulmonary embolism (APE) is a clinical syndrome of pulmonary circulation disorder caused by obstruction of the pulmonary artery or its branches. Histone deacetylase 6 (HDAC6) has been reported to play an important role in lung-related diseases. However, the functional role of HDAC6 in APE remains unclear. Material and methods. Male Sprague Dawley rats were used. The APE model was constructed by inserting an intravenous cannula into the right femoral vein and injecting Sephadex G-50 microspheres (12 mg/kg; 300 μm in diameter). After 1 h, the control and APE rats were intraperitoneally injected with tubastatin A (TubA) (40 mg/kg, an inhibitor of HDAC6) and sampled at 24 h after modeling. H&E staining, arterial blood gas analysis, and wet/dry (W/D) weight ratio were used to evaluate the histopathological changes and pulmonary function in APE rats. ELISA, Western blot, and immunohistochemistry were used to explore the potential mechanism of HDAC6-mediated inflammation in APE. Results. The results indicated that HDAC6 expression was significantly increased in lungs of APE rats. TubA treatment in vivo decreased HDAC6 expression in lung tissues. HDAC6 inhibition alleviated histopathological damage and pulmonary dysfunction, as evidenced by decreased PaO2/FiO2 ratio and W/D weight ratio in APE rats. Furthermore, HDAC6 inhibition alleviated APE-induced inflammatory response. Specifically, APE rats exhibited increased production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-18, however, this increase was reversed by HDAC6 inhibition. Meanwhile, the activation of the NLRP3 inflammasome was also observed in lungs of APE rats, while HDAC6 inhibition blocked this activation. Mechanically, we demonstrated that HDAC6 inhibition blocked the activation of the protein kinase B (AKT)/extracellular signal-regulated protein kinase (ERK) signaling pathway, a classic pathway promoting inflammation. Conclusions. These findings demonstrate that the inhibition of HDAC6 may alleviate lung dysfunction and pathological injury resulting from APE by blocking the AKT/ERK signaling pathway, providing new theoretical fundamentals for APE therapy. [ABSTRACT FROM AUTHOR]

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA's that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones. Microbial production of oleochemicals involves strategies of expressing thioesterase to narrow the substrate pool for the termination enzyme at the expense of one ATP. Here, the authors developed an alternative energy-efficient strategy to use of an acyl-ACP transacylase to produce medium chain oleochemicals in E. coli. [ABSTRACT FROM AUTHOR]

Amides are one of the most fundamental chemical bonds in nature. In addition to proteins and other metabolites, many valuable synthetic products comprise amide bonds. Despite this, there is a need for more sustainable amide synthesis. Herein, we report an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds. This synergistic one-pot combination of chemo- and biocatalysis provides an amide bond disconnection to precursors, that are orthogonal to those in classical amide synthesis, obviating the need for protecting groups and delivering amides in a manner unachievable using existing catalytic regimes. Our integrated approach also affords broad scope, very high (molar) substrate loading, and has excellent functional group tolerance, telescoping routes to natural product derivatives, drug molecules, and challenging chiral amides under environmentally friendly conditions at scale. Proteins, other metabolites and many valuable synthetic products contain amide bonds and there is a need for more sustainable amide synthesis routes. Here the authors show an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) is a very common, severe disease with a lifetime prevalence of ~ 10%. The pathogenesis of MDD is unknown and, unfortunately, therapy is often insufficient. We have previously reported that ceramide levels are increased in the blood plasma of patients with MDD and in mice with experimental MDD. Here, we demonstrate that ceramide-enriched exosomes in the blood plasma are increased in mice with stress-induced MDD. Genetic studies reveal that neutral sphingomyelinase 2 is required for the formation of ceramide-enriched exosomes in the blood plasma. Accordingly, induced deficiency of neutral sphingomyelinase 2 prevented mice from the development of stress-induced MDD. Intravenous injection of microparticles from mice with MDD or injection of ceramide-loaded exosomes induced MDD-like behavior in untreated mice, which was abrogated by ex vivo pre-incubation of purified exosomes with anti-ceramide antibodies or ceramidase. Mechanistically, injection of exosomes from mice with MDD or injection of ex vivo ceramide-loaded microparticles inhibited phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus, which has been previously shown to mediate MDD by plasma ceramide. In summary, our data indicate that ceramide-enriched exosomes are released by neutral sphingomyelinase 2 into the blood plasma upon stress and mediate stress-induced MDD. Key messages: Stress induces ceramide-enriched exosomes in the blood plasma. Ceramide-enriched exosomes mediate major depressive disorder (MDD). Deficiency of neutral sphingomyelinase 2 protects from stress-induced MDD. Neutralization or digestion of ceramide in exosomes prevents stress-induced MDD. Ceramide-enriched exosomes inhibit endothelial phospholipase D in the hippocampus. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is one of the most notorious and ubiquitous soilborne plant pathogens, causing serious economic losses to a large number of hosts worldwide. Although virulence factors have been identified in this filamentous fungus, including various cell-wall-degrading enzymes, toxins, oxalic acids and effectors, our understanding of its virulence strategies is far from complete. To explore novel factors contributing to disease, a new pipeline combining forward genetic screening and next-generation sequencing was utilized in this study. Analysis of a hypovirulent mutant revealed that a mutation in an amidase-encoding gene, Sscle_10g079050, resulted in reduced virulence. This is a first report on the contribution of an amidase to fungal virulence, likely through affecting oxalic acid homeostasis. [ABSTRACT FROM AUTHOR]