MdPRX34L, a class III peroxidase gene, activates the immune response in apple to the fungal pathogen Botryosphaeria dothidea.

MAIN CONCLUSION: MdPRX34L enhanced resistance to Botryosphaeria dothidea by increasing salicylic acid (SA) and abscisic acid (ABA) content as well as the expression of related defense genes. The class III peroxidase (PRX) multigene family is involved in complex biological processes. However, the molecular mechanism of PRXs in the pathogen defense of plants against Botryosphaeria dothidea (B. dothidea) remains unclear. Here, we cloned the PRX gene MdPRX34L, which was identified as a positive regulator of the defense response to B. dothidea, from the apple cultivar 'Royal Gala.' Overexpression of MdPRX34L in apple calli decreased sensitivity to salicylic acid (SA) and abscisic acid（ABA). Subsequently, overexpression of MdPRX34L in apple calli increased resistance to B. dothidea infection. In addition, SA contents and the expression levels of genes related to SA synthesis and signaling in apple calli overexpressing MdPRX34L were higher than those in the control after inoculation, suggesting that MdPRX34L enhances resistance to B. dothidea via the SA pathway. Interestingly, infections in apple calli by B. dothidea caused an increase in endogenous levels of ABA followed by induction of ABA-related genes expression. These findings suggest a potential mechanism by which MdPRX34L enhances plant-pathogen defense against B. dothidea by regulating the SA and ABA pathways.

ortho-Cyanomethylation of aryl fluoroalkyl sulfoxides via a sulfonium-Claisen rearrangement.

We hereby report the ortho-cyanomethylation of aryl fluoroalkyl sulfoxides with acetonitrile through a sulfonium-Claisen-type rearrangement. This reaction enables the incorporation of two valuable functional groups, such as the cyanomethyl group and the fluoroalkylthio group, into arenes. Remarkably, fluoroalkylthio groups, such as SCFH2 and SCF2H, bearing active hydrogen, are well tolerated by the reaction. The success of the reaction relies on the use of an excess amount of acetonitrile and the electronegative effect of fluoroalkyl substituents, both of which promote the electrophilic assembly of sulfoxides with acetonitrile. Consequently, the sulfonium-Claisen rearrangement reaction tolerates a wide variety of fluoroalkyl sulfoxides bearing functional groups including halides, nitriles, ketones, sulfones, and amides, which are appealing for subsequent elaboration and exploration.

Tailored Exfoliation of Polymeric Carbon Nitride for Photocatalytic H2O2 Production and CH4 Valorization Mediated by O2 Activation.

The exfoliation of bulk C3N4 (BCN) into ultrathin layered structure is an effective strategy to boost photocatalytic efficiency by exposing interior active sites and accelerating charge separation and transportation. Herein, we report a novel nitrate anion intercalation-decomposition (NID) strategy that is effective in peeling off BCN into few-layer C3N4 (fl-CN) with tailored thickness down to bi-layer. This strategy only involves hydrothermal treatment of BCN in diluted HNO3 aqueous solution, followed by pyrolysis at various temperatures. The decomposition of the nitrate anions not only exfoliates BCN and changes the band structure, but also incorporates oxygen species onto fl-CN, which is conducive to O2 adsorption and hence relevant chemical processes. In photocatalytic O2 reduction under visible light irradiation, the H2O2 production rate over the optimal fl-CN-530 catalyst is 952 µmol g-1 h-1, which is 8.8 times that over BCN. More importantly, under full arc irradiation and in the absence of hole scavenger, CH4 can be photocatalytically oxidized by on-site formed H2O2 and active oxygen species to generate value-added C1 oxygenates with high selectivity of 99.2 % and record-high production rate of 1893 µmol g-1 h-1 among the metal-free C3N4-based photocatalysts.

The Aux/IAA protein TaIAA15-1A confers drought tolerance in Brachypodium by regulating abscisic acid signal pathway.

KEY MESSAGE: Overexpression of the Aux/IAA protein TaIAA15-1A from wheat improves drought tolerance by regulating the ABA signalling pathway in transgenic Brachypodium. Drought is a major abiotic stress that causes severe crop yield loss. Aux/IAA genes have been shown to be involved in drought stress responses. However, to the best of our knowledge, there has been little research on the molecular mechanism of the wheat Aux/IAA gene in the context of drought tolerance. In this study, we found that expression of the wheat Aux/IAA gene TaIAA15-1A was upregulated by PEG6000, NaCl, SA, JA, IAA and ABA. Transgenic plants overexpressing TaIAA15-1A showed higher drought tolerance than wild-type (WT) plants. The physiological analyses showed that the transgenic lines exhibited a higher survival rate, shoot length, and relative water content than the WT plants. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were enhanced in transgenic lines, causing a reduction in the hydrogen peroxide (H2O2) and superoxide anion radical (O2-) contents. Transcriptome analysis showed that TaIAA15-1A overexpression alters the expression of these genes involved in the auxin signalling pathway, ABA signalling pathway, phenolamides and antioxidant pathways. The results of exogenous ABA treatment suggested that TaIAA15-1A overexpression increased sensitivity to ABA at the germination and postgermination stages compared to WT plants. These results indicate that TaIAA15-1A plays a positive role in plant drought tolerance by regulating ABA-related genes and improving antioxidative stress ability and has potential application in genetically modified crops.

Effect of yeast culture supplementation on blood characteristics, body development, intestinal morphology, and enzyme activities in geese.

The objective of this experiment is to evaluate the effects of yeast culture (YC) supplementation on blood characteristics, body size, carcass characteristics, organ weights, intestinal morphology, and enzyme activities. Five groups of geese were randomly assigned to five dietary treatments: the basal diet (control) and basal diets plus 0.5%, 1.0%, 2.0%, or 4.0% YC. Compared with the controls, YC supplementation at 0.5% and 1.0% increased the serum total protein (TP), albumin (ALB), and globulin (GLO) and decreased the uric acid and creatine kinase (CK) contents (p < 0.05). YC supplementation at 2.0% and 4.0% increased the CK, growth hormone, catalase and glutathione reductase contents, and relative proventriculus weights, and decreased the TP, ALB, and GLO contents, relative liver, gizzard, jejunum, ileum, and thymus weights (p < 0.05). YC supplementation at 2.0% improved fossil bone length, breast muscle percentage, jejunal villus height, ileal and jejunal villus height/crypt depth ratios, pepsin, lipase, amylase and pancreatic trypsin activities, and decreased abdominal fat percentage (p < 0.05). Furthermore, YC inclusion increased the body slope length (linear, p = 0.002; quadratic, p = 0.02), breast width (quadratic, p = 0.02), ileal (linear, p = 0.04; quadratic, p = 0.01) and duodenal villus height (cubic, p = 0.04), and decreased the relative gizzard (quadratic, p = 0.04) and thymus (linear, p = 0.002; quadratic, p = 0.02; cubic, p = 0.02) weights, liver (linear, p = 0.002; quadratic, p = 0.02), and serum (linear, p = 0.006; quadratic, p = 0.03) malondialdehyde contents, and jejunal crypt depth (quadratic, p = 0.03). The findings indicated that the YC supplementation had a positive effect on the growth and development of geese, with 2% YC being the most effective.

Alleviating sample imbalance in water quality assessment using the VAE-WGAN-GP model.

Water resources are essential for sustaining human life and promoting sustainable development. However, rapid urbanization and industrialization have resulted in a decline in freshwater availability. Effective prevention and control of water pollution are essential for ecological balance and human well-being. Water quality assessment is crucial for monitoring and managing water resources. Existing machine learning-based assessment methods tend to classify the results into the majority class, leading to inaccuracies in the outcomes due to the prevalent issue of imbalanced class sample distribution in practical scenarios. To tackle the issue, we propose a novel approach that utilizes the VAE-WGAN-GP model. The VAE-WGAN-GP model combines the encoding and decoding mechanisms of VAE with the adversarial learning of GAN. It generates synthetic samples that closely resemble real samples, effectively compensating data of the scarcity category in water quality evaluation. Our contributions include (1) introducing a deep generative model to alleviate the issue of imbalanced category samples in water quality assessment, (2) demonstrating the faster convergence speed and improved potential distribution learning ability of the proposed VAE-WGAN-GP model, (3) introducing the compensation degree concept and conducting comprehensive compensation experiments, resulting in a 9.7% increase in the accuracy of water quality assessment for multi-classification imbalance samples.

Zearalenone affects the growth of endometriosis via estrogen signaling and inflammatory pathways.

Endometriosis is a chronic, inflammatory, estrogen-dependent gynecological disease characterized by the growth of endometrial stromal cells and glands outside the uterine cavity in response to hormones, which commonly occurs in reproductive-age women. Zearalenone (ZEA) is a toxic metabolite produced by Fusarium, which acts as estrogen activity because of the similarity of its structure to estrogen. In this study, we used an endometriosis mouse model: 15 days after ovariectomy, endometrial fragments were sutured on the pelvic wall, and exogenous estrogen was supplied using an estrogen-releasing silicone tube embedded subcutaneously. Mice were treated with different doses of ZEA by gavage for 21 days. The results show that ZEA significantly inhibited the growth of ectopic endometrium in a dose-dependent manner. The proliferation of cells decreased while apoptosis increased in the ectopic tissues of ZEA-treated mice compared to the vehicle group. The expression of estrogen receptor-α and its downstream targets MUC1 and p-AKT decreased, indicating an impaired estrogen signaling activity by ZEA treatment. In addition, the decreased expression of pro-inflammatory cytokine Tnf-α, Il-1ß, and Il-6, the lower number of macrophages and neutrophils cells, and the inhibited NF-κB signaling pathway suggest the inflammatory response in the ectopic endometrium was also suppressed by ZEA treatment. However, when the exogenous estrogen supply is removed, ZEA, in turn, plays an estrogen-like role that promotes cell proliferation in the ectopic endometrium. In summary, our data suggest ZEA acts as an antagonist in endometriotic tissue when estrogen is sufficient but turns to estrogenic activity in the absence of estrogen in the development of endometriosis. ZEA also inhibits ectopic tissue growth by inhibiting inflammatory response in the endometriosis model.

Morphine promotes tumorigenesis and cetuximab resistance via EGFR signaling activation in human colorectal cancer.

Morphine, a mu-opioid receptor (MOR) agonist, has been extensively used to treat advanced cancer pain. In particular, in patients with cancer metastasis, both morphine and anticancer drugs are given simultaneously. However, evidence showed that morphine might be a risk factor in promoting the tumor's malignant potential. In this study, we report that treatment with morphine could activate MOR and lead to the promotion of proliferation, migration, and invasion in HCT116 and DLD1 colorectal cancer (CRC) cells with time-concentration dependence. Moreover, morphine can also contribute to cetuximab's drug resistance, a targeted drug widely used to treat advanced CRC by inducing the activation of epidermal growth factor receptor (EGFR). The cell phenotype includes proliferation, migration, invasion, and drug resistance, which may be reversed by MOR knockdown or adding nalmefene, the MOR receptor antagonist. Receptor tyrosine kinase array analysis revealed that morphine selectively induced the transactivation of EGFR. EGFR transactivation resulted in the activation of ERK1/2 and AKT. In conclusion, morphine induces the transactivation of EGFR via MOR. It activates the downstream signal pathway AKT-MTOR and RAS-MAPK, increases proliferation, migration, and invasion, and promotes resistance to EGFR inhibitors in a CRC cell line. Furthermore, we verified that EGFR inhibition by cetuximab strongly reversed the protumoral effects of morphine in vitro and in vivo. Collectively, we provide evidence that morphine-EGFR signaling might be a promising therapeutic target for CRC patients, especially for cetuximab-resistant CRC patients.

Domain-adversarial multi-task framework for novel therapeutic property prediction of compounds.

MOTIVATION: With the rapid development of high-throughput technologies, parallel acquisition of large-scale drug-informatics data provides significant opportunities to improve pharmaceutical research and development. One important application is the purpose prediction of small-molecule compounds with the objective of specifying the therapeutic properties of extensive purpose-unknown compounds and repurposing the novel therapeutic properties of FDA-approved drugs. Such a problem is extremely challenging because compound attributes include heterogeneous data with various feature patterns, such as drug fingerprints, drug physicochemical properties and drug perturbation gene expressions. Moreover, there is a complex non-linear dependency among heterogeneous data. In this study, we propose a novel domain-adversarial multi-task framework for integrating shared knowledge from multiple domains. The framework first uses an adversarial strategy to learn target representations and then models non-linear dependency among several domains. RESULTS: Experiments on two real-world datasets illustrate that our approach achieves an obvious improvement over competitive baselines. The novel therapeutic properties of purpose-unknown compounds that we predicted have been widely reported or brought to clinics. Furthermore, our framework can integrate various attributes beyond the three domains examined herein and can be applied in industry for screening significant numbers of small-molecule drug candidates. AVAILABILITY AND IMPLEMENTATION: The source code and datasets are available at https://github.com/JohnnyY8/DAMT-Model. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clinical characteristics of re-hospitalized COVID-19 patients with recurrent positive SARS-CoV-2 RNA: a retrospective study.

At present, it has been noticed that some patients recovered from COVID-19 present a recurrent positive RNA test of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) after being discharged from hospitals. The purpose of the current study was to characterize the clinical features of re-hospitalized patients with recurrent SARS-CoV-2 positive results. From January 12 to April 1 of 2020, our retrospective study was conducted in China. The exposure history, baseline data, laboratory findings, therapeutic schedule, and clinical endpoints of the patients were collected. All the patients were followed until April 10, 2020. Among all COVID-19 patients included in the current study, there were 14 re-hospitalized patients due to recurrent positive tests of SARS-CoV-2 RNA. Fever (11 [78.6%]), cough (10 [71.4%]), and fatigue (7 [50.0%]) were the most common symptoms on the patient's first admission, and less symptoms were found on their second admission. The average duration from the onset of symptoms to admission to hospital was found to be 8.4 days for the first admission and 2.6 days for the second admission (P = 0.002). The average time from the detection of RNA (+) to hospitalization was 1.9 days for the first admission and 2.6 days for the second admission (P = 0.479), and the average time from RNA (+) to RNA (-) was 11.1 days for the first admission and 6.3 days for the second admission (P = 0.030). Moreover, the total time in hospital was 18.6 days for the first admission and 8.0 days for the second admission (P = 0.000). It may be necessary to increase the isolation observation time and RT-PCR tests should be timely performed on multiple samples as soon as possible.

Structural simplification of evodiamine: Discovery of novel tetrahydro-ß-carboline derivatives as potent antitumor agents.

Natural products (NPs) have played a crucial role in the discovery and development of antitumor drugs. However, the high structural complexity of NPs generally results in unfavorable physicochemical profiles and poor drug-likeness. A powerful strategy to tackle this obstacle is the structural simplification of NPs by truncating nonessential structures. Herein, a series of tetrahydro-ß-carboline derivatives were designed by elimination of the D ring of NP evodiamine. Structure-activity relationship studies led to the discovery of compound 45, which displayed highly potent antitumor activity against all the tested cancer cell lines and excellent in vivo antitumor activity in the HCT116 xenograft model with low toxicity. Further mechanistic research indicated that compound 45 acted by dual Top1/2 inhibition and induced caspase-dependent cell apoptosis coupled with G2/M cell cycle arrest. This proof-of-concept study validated the effectiveness of structural simplification in NP-based drug development, discovered compound 45 as a potent antitumor lead compound and enriched the structure-activity relationships of evodiamine.

Expression pattern of mitogen-activated protein kinase kinase 4 and regulation to antibacterial factor ABF-1/2 in response to bacterial challenge from Artemia parthenogenetica.

Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.

Low preoperative prealbumin predicts the prevalence of complications following liver transplantation.

BACKGROUND: As a nutritional index, preoperative serum prealbumin highly correlates with surgical complications. However, the correlation between preoperative prealbumin and postoperative complications remains unclear in liver transplantation (LT). METHODS: A total of 191 patients who underwent LT between 2015 and 2019 were included in the retrospective analysis. According to a cut-off value calculated from a receiver operating characteristic (ROC) curve, the patients were divided into normal and low preoperative prealbumin groups. Univariable and multivariable logistic regression analyses were used to identify independent risk factors for postoperative complications. In addition, patients were divided into subgroups by Model for End-stage Liver Disease (MELD) score, and the association between preoperative prealbumin and postoperative complications was also assessed in each group. RESULTS: A total of 111 (58.1%) patients were included in the low prealbumin group based on a cut-off value of 120 mg/L. The area under the ROC curve (AUC) was 0.754 (95% confidence interval [CI] 0.678-0.832). Low prealbumin (95% CI 1.51-12.8, P = 0.007) was identified as a predictor for postoperative complications based on multivariable regression. In the low and normal prealbumin groups, the prevalence rates of postoperative complications were 27.5% and 8.0% (P = 0.003) in the MELD score ≤ 15 subgroup and 53.3% and 20.0% (P = 0.197) in the MELD score > 15 subgroup, respectively. CONCLUSIONS: Preoperative prealbumin was associated with postoperative complications in LT, and preoperative nutritional support benefitted postoperative recovery, especially for patients with low MELD scores.

An Outbreak of Botulinum Types A, B, and E Associated With Vacuum-Packaged Salted Fish and Ham.

BACKGROUND: Botulinum toxin is a neurotoxin generated during the growth of Clostridium botulinum and can lead to a rare but life-threatening disease with neurologic symptoms. Relatively little is known about the many types of botulinum toxins in China. The clinical symptoms of the different types of botulism toxin-induced illnesses appear after an incubation period and vary greatly by the serotype and degree of exposure to the toxin. CASE REPORT: We describe 4 patients who consumed vacuum-packaged salted fish and ham and then presented with severe gastrointestinal symptoms, resulting in vomiting and one death. Blood serum and urine samples tested by the botulinum neurotoxin Endopep-MS assay were positive for botulinum toxin types A, B, and E. The patient who consumed the largest amount of fish and ham died after 36 h, and the other patients were treated with botulism antitoxin after being diagnosed with a botulinum toxin-induced illness and recovered after 1 month. These cases illustrate the importance of early diagnosis and early treatment of botulism type E, in particular, because of the risk of respiratory failure and treatment delays, which can result in increased mortality and poorer overall outcomes. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Our cases illustrate the importance of early diagnosis and especially the treatment of illnesses related to botulism type E because of the risk of respiratory failure and because treatment delays can result in increased mortality and worsened overall outcomes.

Temporal dynamics of Arc/Arg3.1 expression in the dorsal striatum during acquisition and consolidation of a motor skill in mice.

Region- and pathway-specific plasticity within striatal circuits is critically involved in the acquisition and long-term retention of a new motor skill as it becomes automatized. However, the molecular substrates contributing to this plasticity remain unclear. Here, we examined the expression of the activity-regulated cytoskeleton-associated protein (Arc) in the associative or dorsomedial striatum (DMS) and the sensorimotor or dorsolateral striatum (DLS), as well as in striatonigral and striatopallidal neurons, during different skill learning phases in the accelerating rotarod task. We found that Arc was mainly expressed in the DMS during early motor learning and progressively increased in the DLS during gradual motor skill consolidation. Moreover, Arc was preferentially expressed in striatopallidal neurons early in training and gradually increased in striatonigral neurons later in training. These data demonstrate that in the dorsal striatum, the expression of Arc exhibits a region- and cell-specific transfer during the learning of a motor skill, suggesting a link between striatal Arc expression and motor skill learning in mice.

Comparative analysis of two brine shrimps revealed differential expression pattern and functional characterization of CK2α under bacterial stimulation from different geographical distribution.

Understanding how the brine shrimp responds to different geographical populations can provide novel insights on response to bacterial stimulation. In the paper, Artemia sinica from lower altitudes and Artemia parthenogenetica from higher altitudes of the Tibetan Plateau, were used to illustrate different defense against bacteria mechanisms that these organisms used to adapt to different geographical environments. Protein kinase CK2 is a serine/threonine kinase with a multitude of protein substrates. It is a ubiquitous enzyme essential for the viability of eukaryotic cells, where its functions in a variety of cellular processes, including cell cycle progression, apoptosis, transcription, and viral infection. The gene encodes the same mRNA sequence in A. sinica and A. parthenogenetica, named AsCK2α and ApCK2α, respectively. The open reading frame was obtained, a 1047-bp sequence encoding a predicted protein of 349 amino acids. To systematically analyze the expression of AsCK2α and ApCK2α during embryonic development and bacterial challenge, real-time PCR, Western blotting and immunohistochemistry were performed. The results showed that AsCK2α was higher than ApCK2α at different developmental stages. Under bacterial challenge, the expression of ApCK2α was significantly higher than AsCK2α. Protein localization analysis showed that AsCK2α and ApCK2α were mainly distributed in the head and chest. Our research revealed that CK2α plays a vital role in the growth, development and bacterial stimulation of the brine shrimp.

High-throughput sequencing reveals biofluid exosomal miRNAs associated with immunity in pigs.

Large numbers of miRNAs are found in biofluid exosomes. We isolated ~50-200 nm diameter exosomes from four types of porcine biofluid (urine, plasma, semen, and bile) using serial centrifugation and ultracentrifugation procedures. A total of 42.15 M raw data were generated from four small RNA libraries. This produced 40.17 M map-able sequences, of which we identified 204 conserved miRNAs, and 190 novel candidate miRNAs. Furthermore, we identified 34 miRNAs specifically expressed in only one library, all with well-characterized immune-related functions. A set of five universally abundant miRNAs (miR-148a-3p, miR-21-5p, let-7f-5p, let-7i-5p, and miR-99a-5p) across all four biofluids was also found. Function enrichment analysis revealed that the target genes of the five ubiquitous miRNAs are primarily involved in immune and RNA metabolic processes. In summary, our findings suggest that porcine biofluid exosomes contain a large number of miRNAs, many of which may be crucial regulators of the immune system.

Profiling the microbial contamination in aviation fuel from an airport.

Microbial contamination during fuel storage can cause fuel system fouling and corrosion. Characterizing microbial contamination is critical for preventing and solving these problems. In this study, culture-based combing with the culture-independent methods, were used to profile the microbial contamination in aviation fuel. High-throughput sequencing (HTS) modified by propidium monoazide (PMA) revealed a higher diversity of contaminating microorganisms in samples than the culture method. Proteobacteria (47%), Actinobacteria (21%) and Ascomycota (>99%, fungi) were the most abundant phyla, and the neglected archaea was also detected. Additionally, qPCR-based methods revealed all samples contained a heavy level of microbial contamination, which was more accurate than its culturable counterparts, and fungal contamination was still a problem in aviation fuel. The application of a PCR-based method gives deeper insight into microbial contamination in aviation fuel than the conventional culture method, thus using it for regular detection and accurate description of fuel contamination is strongly recommended in the case of explosive microbial growth.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa.

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ±â€¯0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ±â€¯4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ±â€¯0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ±â€¯0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass.

Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.