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Phenazine-based small molecules are nitrogen-containing heterocyclic compounds with diverse bioactivities and electron transfer properties that exhibit promising applications in pharmaceutical and electrochemical industries. However, the biosynthetic mechanism of highly substituted natural phenazines remains poorly understood. In this study, we report the direct cloning and heterologous expression of the lomofungin biosynthetic gene cluster (BGC) from Streptomyces lomondensis S015. Reconstruction and overexpression of the BGCs in Streptomyces coelicolor M1152 resulted in eight phenazine derivatives including two novel hybrid phenazine metabolites, and the biosynthetic pathway of lomofungin was proposed. Furthermore, gene deletion suggested that NAD(P)H-dependent oxidoreductase gene lomo14 is a nonessential gene in the biosynthesis of lomofungin. Cytotoxicity evaluation of the isolated phenazines and lomofungin was performed. Specifically, lomofungin shows substantial inhibition against two human cancer cells, HCT116 and 5637. These results provide insights into the biosynthetic mechanism of lomofungin, which will be useful for the directed biosynthesis of natural phenazine derivatives.

Phenazines are aromatic compounds with antifungal and cytotoxic activities. Phenazines incorporating phenazine 1-carboxylic acid have widespread applications in agriculture, medicine, and industry. Griseoluteic acid is a cytotoxic compound secreted by Streptomyces griseoluteus P510, displaying potential medical applications. However, the biosynthetic pathway of griseoluteic acid has not been elucidated, limiting its development and application. In this study, a conserved phenazine biosynthetic gene cluster of S. griseoluteus P510 was identified through genomic analysis. Subsequently, its was confirmed that the four essential modification enzymes SgpH, SgpI, SgpK, and SgpL convert phenazine-1,6-dicarboxylic acid into griseoluteic acid by heterologous expression in Escherichia coli . Moreover, the biosynthetic pathway of griseoluteic acid was established in Pseudomonas chlororaphis characterized by a high growth rate and synthesis efficiency of phenazines, laying the foundation for the efficient production of griseoluteic acid.

Polyhydroxyalkanoates (PHAs) are promising alternatives to petroleum-based plastics, owing to their biodegradability and superior material properties. Here, the controllable biosynthesis of scl- co -mcl PHA containing 3-hydroxybutyrate (3HB) and mcl 3-hydroxyalkanoates was achieved in Pseudomonas chlororaphis HT66. First, key genes involved in fatty acid β-oxidation, the de novo fatty acid biosynthesis pathway, and the phaC1 - phaZ - phaC2 operon were deleted to develop a chassis strain. Subsequently, an acetoacetyl-CoA reductase gene phaB and a PHA synthase gene phaC with broad substrate specificity were heterologously expressed for producing and polymerizing the 3HB monomer with mcl 3-hydroxyalkanoates under the assistance of native β-ketothiolase gene phaA . Furthermore, the monomer composition of scl- co -mcl PHA was regulated by adjusting the amount of glucose and dodecanoic acid supplemented. Notably, the cell dry weight and scl- co -mcl PHA content reached 14.2 g/L and 60.1 wt %, respectively, when the engineered strain HT11Δ:: phaCB was cultured in King's B medium containing 5 g/L glucose and 5 g/L dodecanoic acid. These results demonstrated that P. chlororaphis can be a platform for producing scl- co -mcl PHA and has the potential for industrial application.

The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism Pseudomonas chlororaphis is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes lacZ encoding β-galactosidase from Escherichia coli K12 and phzO encoding PCA monooxygenase from P. chlororaphis GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed P MOK_RS0128085 UTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity P MOK_RS0116635 UTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for P. chlororaphis , providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia., Significance: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Polycystic ovary syndrome (PCOS) was known as the common endocrine disease in women, featured as hyperandrogenism, ovulation disorders, etc. Fat mass and obesity-associated protein (FTO), a m6A demethylase, is abnormal in the occurrence of ovarian diseases. However, the mechanism of FTO in the pathogenesis of PCOS is still unclear., Methods: The level of FTO in clinical samples, PCOS rat with hyperandrogenism and granulosa cells (GCs) lines effected by DHT were investigated by ELISA, qRT-PCR, WB, and IHC, while m6A RNA methylation level was studied by m6A Colorimetric and androgen level was tested through ELISA. Changes in steroid hormone synthetase and androgen receptor (AR)/prostate-specific antigen (PSA) levels in vitro were visualized by WB after transient transfection silenced FTO. The effect of DHT combined with FTO inhibitor meclofenamic acid (MA) on FTO, AR/PSA, and AKT phosphorylation were also demonstrated by WB. The co-localization of FTO and AR in KGN cells was analyzed by confocal microscopy, and the physiological interaction between FTO and AR was studied by Co-IP assay. The effect of FTO-specific inhibitor MA, AKT phosphorylation inhibitor LY294002, and the combined them on GCs proliferation and cell cycle were evaluated by drug combination index, EDU assay, and flow cytometry analysis., Results: FTO expression was upregulated in follicular fluid and GCs in PCOS patients clinically. The high FTO expression in patients was negative with the level of m6A, but positive with the level of androgen. The upregulation of FTO was accompanied with a decrease in the level of m6A in PCOS rat with hyperandrogenism. Dihydrotestosterone (DHT) promoted the FTO expression and inhibited m6A content as a dose-dependent way in vitro . In contrast, suppression of FTO with siRNA attenuated the expression of steroid hormone synthetase such as CYP11A1, CYP17A1, HSD11B1, HSD3B2 except CYP19A1 synthetase, ultimately inducing the decrease of androgen level. Suppression of FTO also decreased the biological activity of androgen through downregulation AR/PSA. MA treatment as the specific FTO antagonist decreased cell survival in time- and dose-dependent way in GCs lines. Correspondingly, MA treatment decreased the expression of FTO, AR/PSA expression, and AKT phosphorylation in the presence of DHT stimulation. Additionally, we also speculate there is a potential relation between FTO and AR according to FTO was co-localized and interacted with AR in KGN cells. Compared with AKT phosphorylation inhibitor LY294002 or MA alone, LY294002 combined with MA synergistically inhibited cell survival and increased G2/M phase arrest in GC line., Conclusions: We first evaluated the correlation of FTO and m6A in PCOS clinically, and further explored the mechanism between FTO and hyperandrogenism in PCOS animal and cell models. These findings contributed the potential therapy by targeting the FTO for hyperandrogenism in PCOS.

Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis . Through testing the minimum medium commonly used by Pseudomonas , an ME medium for P. chlororaphis with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe 2+ and NH 4 + on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy.

Objectives: To assess the association of whole grain consumption with the risk of incident knee OA., Material and Methods: We followed 2846 participants in the Osteoarthritis Initiative ages 45-79 years. Participants were free from radiographic knee OA (Kellgren-Lawrence grade <2) in at least one knee at baseline. Dietary data from baseline were obtained using the Block Brief Food Frequency Questionnaire. We defined radiographic knee OA incidence as a Kellgren-Lawrence grade ≥2 during the subsequent 96 months. Cox proportional hazards models were used to assess the association between whole grain food intake and the risk of incident knee OA., Results: During the 96 month follow-up, 518 participants (691 knees) developed incident radiographic knee OA. Higher total whole grain consumption was significantly associated with a lower knee OA risk [hazard ratio (HR)quartile 4vs1 = 0.66 (95% CI 0.52, 0.84), P for trend < 0.01] after adjusting for demographic and socio-economic factors, clinical factors and other dietary factors related to OA. Consistently, a significant inverse association of dark bread consumption with knee OA risk was observed [HRquartile 4vs1 = 0.68 (95% CI 0.53, 0.87), P for trend < 0.01). In addition, we observed a significant inverse association between higher cereal fibre intake and reduced knee OA risk [HRquartile 4vs1 = 0.61 (95% CI 0.46, 0.81), P for trend < 0.01)., Conclusions: Our findings revealed a significant inverse association of whole grain consumption with knee OA risk. These findings provide evidence that eating a diet rich in whole grains may be a potential nutritional strategy to prevent knee OA., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Human chorionic gonadotropin (hCG) might affect endometrial receptivity, exerting integral roles in embryo implantation. This study explored the action of hCG in endometrial receptivity via the miR-126-3p/PIK3R2/PI3K/Akt/eNOS axis. The embryo implantation dysfunction (EID) mouse models were established by administrating mifepristone and human endometrial epithelial cells (EECs) were used for in vivo experiments, both followed by hCG treatment. Expression level of CD105 and protein levels of cadherin CD144 and CD146 in mice were determined by immunohistochemistry and Western blot. The levels of miR-126-3p and PIK3R2 mRNA and PIK3R2, p-PI3K p85 α, PI3K p110 α, p-Akt, Akt, p-eNOS, and eNOS protein levels were measured. Cell proliferation was evaluated by CCK-8 and EdU assays. The binding sites of miR-126-3p and PIK3R2 were predicted and verified. hCG-treated EECs were further transfected with miR-126-inhibitor for functional rescue experiments. hCG ameliorated endometrial receptivity in EID mice. Moreover, hCG promoted miR-126-3p and suppressed PIK3R2 in EID mice and EECs. miR-126-3p targeted PIK3R2. EEC proliferation was enhanced after hCG treatment but inhibited by miR-126-3p downregulation. Both in vivo and in vitro experiments validated that hCG activated the PI3K/Akt/eNOS pathway through the miR-126-3p/PIK3R2 axis. Collectively, hCG improves endometrial receptivity by activating the PI3K/Akt/eNOS pathway via regulating miR-126-3p/PIK3R2., (© 2023 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor β1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Our research question was to evaluate the chromosome concordance of trophectoderm (TE) biopsy with noninvasive chromosome screening (NICS) using embryo culture medium renewed twice on Day 3 (D3) and Day 4 (D4). In this study, we evaluated 64 cycles with 223 biopsied blastocysts. These were categorized into two groups based on replacing embryo culture medium on D3 (control group) or on D3 and D4 (experimental group). The fundamental characteristics and main outcomes were compared. The concordance rates of NICS results with TE biopsy were determined according to next generation sequencing results. In total, 103 experimental and 120 control embryo cultures were collected, and the euploid status was analyzed using NICS technology. The overall concordance rates with TE biopsy of the experimental and control groups were 0.86 and 0.75, respectively. Statistically significant difference was found between the two groups. An additional medium renewal of the D4 embryo culture can improve the concordance of NICS with TE biopsy.

Infertility is a major health concern worldwide. This retrospective study aimed to assess the predictive value of the morphokinetic parameters of temporary-arrest embryos for the pregnancy outcomes of women undergoing frozen embryo transfer (FET) cycles. In this study, we evaluated 244 FET cycles with 431 day-4 temporary-arrest embryos. They were categorized into two groups (pregnancy and non-pregnancy) according to the pregnancy outcomes of the women after embryo transfer on day 5, and their fundamental characteristics were compared. The morphokinetic parameters from the time-lapse monitoring system were assessed according to different pregnancy outcomes. The mean number of embryo blastomeres thawed on day 3 in the pregnancy group was 7.47, which was significantly higher than the number in the non-pregnancy group ( p < 0.01). Besides, embryos in the non-pregnancy group contained more embryo fragments and lower grades than those in the pregnancy group ( p < 0.001). Furthermore, morphokinetic parameters: tPNa, t2, t5, and t5&#95;tPNf showed a statistical difference between the pregnancy and non-pregnancy groups ( p < 0.05). Receiver-operating characteristic analysis revealed that the time from pronuclear fading to the 5-cell stage (t5&#95;PNF) predicted the clinical prognosis outcomes (area under the curve = 0.64; 95% confidence interval [CI], 0.58-0.70; p < 0.001). The morphokinetic parameter t5&#95;PNF could be regarded as a potential implantation predictor in our study., Competing Interests: Conflict of interest: There are no conflict of interest., (© 2022 the author(s), published by De Gruyter.)

Aims: Phenazines, such as phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA), 2-hydroxyphenazine (2-OH-PHZ), are a class of secondary metabolites secreted by plant-beneficial Pseudomonas. Ps. chlororaphis GP72 utilizes glycerol to synthesize PCA, 2-OH-PCA and 2-OH-PHZ, exhibiting broad-spectrum antifungal activity. Previous studies showed that the addition of dithiothreitol (DTT) could increase the phenazines production in Ps. chlororaphis GP72AN. However, the mechanism of high yield of phenazine by adding DTT is still unclear., Methods and Results: In this study, untargeted and targeted metabolomic analysis were adopted to determine the content of metabolites. The results showed that the addition of DTT to GP72AN affected the content of metabolites of central carbon metabolism, shikimate pathway and phenazine competitive pathway. Transcriptome analysis was conducted to investigate the changed cellular process, and the result indicated that the addition of DTT affected the expression of genes involved in phenazine biosynthetic cluster and genes involved in phenazine competitive pathway, driving more carbon flux into phenazine biosynthetic pathway. Furthermore, genes involved in antioxidative stress, phosphate transport system and mexGHI-opmD efflux pump were also affected by adding DTT., Conclusion: This study demonstrated that the addition of DTT altered the expression of genes related to phenazine biosynthesis, resulting in the change of metabolites involved in central carbon metabolism, shikimate pathway and phenazine competitive pathway., Significance and Impact of the Study: This work expands the understanding of high yield of phenazine by the addition of DTT and provides several targets for increasing phenazine production., (© 2022 Society for Applied Microbiology.)

In order to improve the mechanical strength and imprinting efficiency, a novel bovine serum albumin (BSA) molecularly imprinted poly(ionic liquid)/calcium alginate composite cryogel membrane (MICM) was prepared. The results of the tensile test indicated that the MICM had excellent mechanical strength which could reach up to 90.00 KPa, 30.30 times higher than the poly (ionic liquid) membrane without calcium alginate; the elongation of it could reach up to 93.70%, 8.28 times higher than the poly (ionic liquid) membrane without calcium alginate. The MICM had a very high welling ratio of 1026.56% and macropore porosity of 62.29%, which can provide effective mass transport of proteins. More remarkably, it had a very high adsorption capacity of 485.87 mg g -1 at 20 °C and 0.66 mg mL -1 of the initial concentration of BSA. Moreover, MICM also had good selective and competitive recognition toward BSA, exhibiting potential utility in protein separation. This work can provide a potential method to prepare the protein-imprinted cryogel membrane with both high mechanical strength and imprinting efficiency.

Objective: Sepsis, showing high mortality, is a lethal dysfunction of organs caused by an infection-induced disorder in the host response. It has complicated pathogenesis, which has not yet been elucidated completely. Recently, the principal factors causing pathogenesis and even death in sepsis patients are imbalance in inflammatory response and immunosuppression occurring when the host is challenged by infection. Previous studies found that Lycium barbarum polysaccharide (LBP) worked well in enhancing immunity. This study aims at exploring the efficacy of pretreatment with LPB in regulating splenic immunity during the pathogenesis of sepsis induced by cecum ligation perforation (CLP) in rats., Methods: This research established the cecum ligation perforation rat model. Using immunohistochemistry and flow cytometry, the effects of Lycium barbarum polysaccharide in various doses in influencing splenic immunity and prognosis of sepsis induced by cecum ligation perforation in rats were examined., Results: This study showed that LBP lowered the 72-hour mortality of sepsis rats induced by CLP, relieved systemic inflammation, improved the ratio of T-cell subgroups positive in CD3+, CD4+, or CD8+ and expression of HLA-DR protein, and repaired damage to splenic tissue, implying its efficacy in enhancing the immunity of sepsis rats induced by CLP., Conclusions: LBP may ameliorate clinical symptoms of rats with cecum ligation perforation, improve cellular immunity in the spleen, and treat sepsis so as to provide a theoretical basis for the pathogenesis and development of sepsis as well as its diagnosis and treatment, and offer scientific proof for the development and utilization of LBP applied to critical diseases., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Huan Ding et al.)

We aimed to investigate the association between either or both of benzodiazepines (BZDs) and non-BZDs and the incidence of atrial fibrillation (AF) in the Taiwan National Health Insurance Database. The participants with at least two prescriptions of BZDs and/or non-BZDs were identified as hypnotics users, whereas those without any prescription of hypnotics were non-hypnotics users. The hypnotics and non-hypnotics cohorts were 1:1 matched on their propensity scores. A total of 109,704 AF-free individuals were included; 610 AF cases occurred in the 54,852 hypnotics users and 166 in the 54,852 non-hypnotics users during the 602,470 person-years of follow-up, with a higher risk of new-onset AF in the users than the non-users (hazard ratio (HR): 3.61, 95% confidence interval [CI]: 3.04−4.28). The users at the highest tertiles of the estimated defined daily doses per one year (DDD) had a greater risk for AF than the non-users, with the risk increasing by 7.13-fold (95% CI: 5.86−8.67) for >0.74-DDD BZDs, 10.68-fold (95% CI: 6.13−18.62) for >4.72-DDD non-BZDs, and 3.26-fold (95% CI: 2.38−4.47) for > 1.65-DDD combinations of BZDs with non-BZDs, respectively. In conclusion, hypnotics use was associated with elevated incidence of AF in the Taiwanese population, which highlighted that the high-dose usage of hypnotics needs more caution in clinical cardiological practice.

Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp D10A ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34 + stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34 + stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34 + CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34 + cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34 + cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34 + cells. These data challenged the dependency of OXPHOS for CML CD34 + cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML., (© 2022. The Author(s).)

Mupirocin, a polyketide antibiotic produced by Pseudomonas fluorescens, is used as a topical antimicrobial treatment to cure various skin infections. Quorum sensing system plays an important role in regulation of mupirocin biosynthesis in P. fluorescens NCIMB 10586. In Pseudomonas, the RpeA/RpeB two-component signal transduction (TCST) system regulates quorum sensing system. However, the influences of the RpeA/RpeB TCST system on mupirocin production or other cell activities have not been studied. In this work, the homologous genes of rpeA and rpeB in P. fluorescens NCIMB 10586 were identified and inactivated in the chromosome, respectively. The deletion of rpeA reduced the mupirocin production from 160 in the wild-type to 21.3 mg/L along with slightly decreased cell growth, while no significant effected on mupirocin production in the rpeB mutant. Next, it was found that the RpeA/RpeB TCST system regulated the biosynthesis of mupirocin by modulating the quorum sensing system. Furthermore, untargeted metabolomics analysis was employed to detect the influences of RpeA on other cell activities modulated by quorum sensing system. Combined with quantitative real-time PCR, the results demonstrated that RpeA also regulated other cell activities including central carbon, amino acids, fatty acids, and purine metabolism. Overall, this study expands the current understanding of the RpeA/RpeB TCST system and provides several targets for increasing yields of mupirocin. KEY POINTS: • In P. fluorescens, the RpeA/RpeB TCST system regulates the biosynthesis of mupirocin. • RpeA modulates the cell activities through effecting the central carbon metabolism., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Soluble microbial products (SMP), a type of polymers released from microbial metabolism and decay, show great potential for wastewater treatment as bioflocculants; however, biogenic flocculant utilization is currently limited to bacterial SMP. In this study, SMP produced by Phanerochaete chrysosporium BKMF-1767 (SMP-P) was investigated to determine the application potential of fungal SMP. SMP-P exhibited high flocculation activity in kaolin suspension at a dosage range of 0.67-0.84 mg/L with Ca 2+ assistance, comparable to that of commercial polyacrylamide. The high molecular weight polysaccharides (2.0 × 10 6 -4.7 × 10 7  Da) in SMP-P, which enabled flocculation via the bridging mechanism and served as the dominant active constituent, were composed of glucose and arabinose at a molar ratio of 1: 0.03, with (1 → 4, 6)-linked glucose as the main backbone and a small proportion of branched structures. They contained hydroxyl and carboxyl, effective functional groups for the flocculation process, and displayed parallel self-orientation behavior in water. Efficient chemical oxygen demand removal was achieved during municipal wastewater treatment using SMP-P as the bioflocculant. This study demonstrates the feasibility of utilizing fugal SMP as bioflocculants and provides guidance for their practical application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The natural-product-based low-molecular-weight supramolecular hydrogels (LMWSHs) induced by heating are rarely reported. In this work, a simple salt of oleanolic acid (OA) and choline ([choline][OA]) was used as the natural product hydrogelator (NPHG) to form LMWSHs. Unlike common sol-gel transitions, the OA-based LMWSH displayed a unique property with which the system could undergo a phase transition from the sol state to the gel state upon heating. Moreover, the phase separation was observed in sol and gel states when the temperature was elevated with nonreversible transparent-turbid transitions. LMWSHs showed good stability and injectability and the potential to be a drug delivery vehicle for sustained release of drugs. In this regard, this work provided a facile approach to designing an OA-based NPHG for preparing heat-induced LMWSHs.

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in Pseudomonas chlororaphis HT66. The phzE , fadA , and fadB genes were deleted to block the native phenazine pathway and weaken the fatty acid β-oxidation pathway. Additionally, a PHA depolymerase gene phaZ was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ:: C1C2J exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.

Objective: To compare clinical application of 1.5 T MRI in acute rotator interval injury., Methods: Totally 160 patients with acute rotator cuff tear by clinical diagnosis were retrospectively analyzed by MRI examination and arthroscopy from March 2016 to February 2019, including 122 males and 38 females, aged from 22 to 71 years old with an average of (42.35±3.48) years old. Based on the results of arthroscopy as the gold standard, the shape and signal changes of rotator cuff, rotator interval, peripheral bursa, bone and soft tissue were observed by MRI on axial, oblique coronal and oblique sagittal imagese., Results: The direct MRI signs of acute rotator interval injury displayed thickening, diminution, distortion, interruption of the coracohumeral ligament and superior glenohumeral ligament complex with highsignal intensity on fat-suppression by proton weighted sequence. The indirect MRI signs displayed rotator cuff, peripheral bone and soft tissue injury. The consistency of the results between the two methods was quite satisfactory (Kappa=0.85), and the concordance rate of the two methods has statistically significant ( Z =10.75, P <0.00). There was no statistical difference in the inconsistent parts between two methods ( χ 2 = 0.400, P >0.05). The true positive rate, true negative rate, false positive rate and false negative rate of MRI diagnosis of rotator interval injury were 93.75%, 91.30%, 94.74%, 5.26%, 8.70% respectively., Conclusion: MRI could clearly display acute rotator interval, and could accurately diagnose acute rotator interval injury, which provide more accurate imaging basis for clinical diagnosis and treatment.

Natural phenazines are a class of multifunctional secondary metabolites of bacteria that play an important role in the biocontrol of plant pathogens. In this paper, a novel bioactive phenazine derivative was isolated from Streptomyces lomondensis S015 through silica gel chromatography and preparative high-performance liquid chromatography (HPLC). The structure was identified as 1-carboxyl-6-formyl-4,7,9-trihydroxy-phenazine (CFTHP) by NMR spectroscopy in combination with ultraperformance liquid chromatography & mass spectrometry (UPLC-MS). CFTHP could inhibit Pythium ultimum , Rhizoctonia solani , Septoria steviae , and Fusarium oxysporum f. sp. niveum with minimal inhibitory concentration (MIC) values of 16, 32, 16, and 16 μg/mL, respectively. A global regulatory gene phoP could positively regulate CFTHP biosynthesis since its production was 3.0-fold enhanced by phoP overexpression and inhibited by phoP deletion in Streptomyces lomondensis S015. These studies illustrated the potential of CFTHP as a promising biopesticide and provided a reference for phenazine production improvement.

Leukemia arises from blockage of the differentiation/maturation of hematopoietic progenitor cells at different stages with uncontrolled proliferation of leukemic cells. However, the signal pathways that block cell differentiation remain unclear. Herein we found that SUMOylation of the M2 isoform of pyruvate kinase (PKM2), a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate, is prevalent in a variety of leukemic cell lines as well as primary samples from patients with leukemia through multiple-reaction monitoring based targeted mass spectrometry analysis. SUMOylation of PKM2 lysine 270 (K270) triggered conformation change from tetrameric to dimeric of PKM2, reduced PK activity, and led to nuclear translocation of PKM2. SUMO1 modification of PKM2 recruits and promotes degradation of RUNX1 via a SUMO-interacting motif, resulting in blockage of myeloid differentiation of NB4 and U937 leukemia cells. Replacement of wild type PKM2 with a SUMOylation-deficient mutant (K270R) abrogated the interaction with RUNX1, and the blockage of myeloid differentiation in vitro and in xenograft model. Our results establish PKM2 as an essential modulator of leukemia cell differentiation and a potential therapeutic target, which may offer synergistic effect with differentiation therapy in the treatment of leukemia.

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.

2-Hydroxyphenazine (2-OH-PHZ) is an effective biocontrol antibiotic secreted by Pseudomonas chlororaphis GP72AN and is transformed from phenazine-1-carboxylic acid (PCA). PCA is the main component of the recently registered biopesticide "Shenqinmycin". Previous research showed that 2-OH-PHZ was better in controlling wheat take-all disease than PCA; however, 2-OH-PHZ production was low under natural conditions. Herein, we confirmed that PCA induced reactive oxygen species in its host P. chlororaphis GP72AN and that the addition of DTT improved PCA production by 1.8-fold, whereas the supplementation of K 3 [Fe(CN) 6 ] and H 2 O 2 increased the conversion rate of PCA to 2-OH-PHZ. Finally, a two-stage fermentation strategy combining the addition of DTT at 12 h and H 2 O 2 at 24 h enhanced 2-OH-PHZ production. Taken together, the two-stage fermentation strategy was designed to enhance 2-OH-PHZ production for the first time, and it provided a valuable reference for the fermentation of other antibiotics.

Background Endometrial cancer (EC) is one of the most common gynaecological tumours in the worldwide. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) promotes cell proliferation, migration and invasion in EC cells. However, the molecular mechanisms of NEAT1 in EC have not been fully clarified. We conducted this study to reveal the function of NEAT1 in EC tissues and cell lines. Materials and methods Cancer and adjacent tissues were collected from EC patients. HEC-1A and Ishikawa cells were cultured in vitro. NEAT1 expression was downregulated by transfecting small hairpin RNA (shRNA) and miR-144-3p was overexpressed by transfecting miR-144-3p mimics. Cell proliferation was detected by MTT assay and colony formation assay. Cell migration and invasion abilities were assessed by transwell assay. A dual-luciferase reporter assay was used to verify the relationship among NEAT1, EZH2, and miR-144-3p. The expression level of EZH2 was measured by Western blot and qPCR. Results NEAT1 was highly expressed in EC tissues and cells. Knockdown of NEAT1 inhibited the proliferation, migration and invasion of EC cells. Additionally, NEAT1 acted as a ceRNA of miR-144-3p, leading to EZH2 upregulation. Overexpression of miR-144-3p suppressed the proliferation and invasion of EC cells. Conclusions NEAT1 promotes EC cells proliferation and invasion by regulating the miR-144-3p/EZH2 axis.

Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical, and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first tim, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp. NRRL12068 (encoded by bomO) and Streptomyces chartreusis NRRL3882 (encoded by calB 3 ) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB 3 was selected for the synthesis of cinnabarinic acid due to its high DHHA conversion rate. Subsequently, cinnabarinic acid was synthesized by the expression of the DHHA dehydrogenase CalB 3 and the phenoxazinone synthase CotA in the DHHA-producing strain P. chlororaphis GP72, resulting in a cinnabarinic acid titer of 20.3 mg/L at 48 hr. Further fermentation optimization by the addition of Cu 2+ , H 2 O 2 , and with adding glycerol increased cinnabarinic acid titer to 136.2 mg/L in shake flasks. The results indicate that P. chlororaphis GP72 may be engineered as a microbial cell factory to produce cinnabarinic acid or its derivatives from renewable bioresources., (© 2019 Wiley Periodicals, Inc.)

Premature ovarian insufficiency is characterized by reduced ovarian function in a young woman, resulting in an earlier menopause compared with women with normal ovarian function. It is a term that reflects the variable nature of the condition and is substantially less emotive than the formerly used 'premature ovarian failure', which suggested a single event in time. Tripterygium glycosides can damage ovarian function and cause infertility. This case report describes a 33-year-old female patient (gravida 0, parity 0) who presented with premature ovarian insufficiency after being treated with tripterygium glycosides for nephrotic syndrome. The woman received oestrogen-progestin replacement therapy (Femoston) and ovulation induction, which resulted in a successful pregnancy. The patient delivered a healthy baby girl. This case demonstrates that it is possible for a woman with tripterygium glycoside-induced premature ovarian insufficiency to become pregnant with the correct therapy.

The effects of P supplementation on chromium(VI) uptake by Leersia hexandra Swartz were studied using pot-culture experiment. P-deficiency and zero-P addition controls were included. The Cr(VI) uptake followed Michaelis-Menten kinetics. Compare with the control, the P-supply decreased the Michaelis constant (Km) by 16.9% and the P-deficiency decreased the maximum uptake velocity (V max ) by 18%, which indicated no inhibition and competition between P and Cr(VI) uptake by L. hexandra. Moreover, there were a synergistic action between P and Cr(VI) suggests that Cr(VI) uptake by the roots of L. hexandra may be an active process. The bioconcentration factor (BCF) and the transport factor (TF') increased with the increase in P supply. The highest BCF was 3.6-folds higher than the control, indicating that the additional P contribute to a higher ability of L. hexandra transporting Cr from root to the aboveground parts.

Digoxin is a member of cardiac glycosides and recent studies show that digoxin plays anticancer role in several types of cancer. However, the anticancer effects and mechanism of digoxin in leukemia is largely unknown. Her, our data show that digoxin treatment significantly inhibits leukemia cell viability. In addition, digoxin treatment significantly induced apoptosis and G2/M cell cycle arrest in leukemia cells. Furthermore, we demonstrated that digoxin treatment inactivate that oncogenic pathway Akt/mTOR signaling in leukemia cells. In addition, our data show that digoxin treatment induces activation of unfolded protein response (UPR) signaling in leukemia cells. Interestingly, our in vitro and in vivo experiments show that combination treatment of digoxin and UPR inhibitor can synergistically suppress leukemia growth and induces apoptosis and cell cycle arrest compared to single drug treatment. In summary, our findings indicate that digoxin has potential anticancer effects on leukemia. The combination of digoxin and UPR signaling inhibitor can exerts synergistic anticancer activity against leukemia. © 2017 BioFactors, 43(6):812-820, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na + ,K + -ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na + ,K + -ATPase α1 in K562 cells, and significantly arrested cells in G 2 /M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML., Competing Interests: None.

Involvement of genes (CYS-A1, CYS-C1 and NIT4) encoded with cysteine synthase, β-cyanoalanine synthase, nitrilase and cyanide metabolisms are evident in Arabidopsis. In the present study, identifications of CYS-A1, CYS-C1 and NIT4, predictions of conserved motifs, and constructions of phylogenetic relationships, based on their amino acid sequences in rice, were conducted. In order to elucidate the transcriptional responses of these cyanide-degrading genes, two candidate homologues were selected for each gene to test their expression changes upon exposure to exogenous KCN in rice seedlings using RT-PCR. Results showed that all selected candidate homologous genes were differentially expressed at different exposure points in roots and shoots of rice seedlings, suggesting their distinct roles during cyanide assimilation. Both candidate homologues for CYS-A1 constantly exhibited more abundant transcripts in comparison to control. However, only one candidate homologue for CYS-C1 and NIT4 showed a remarkable up-regulation during KCN exposure. Analysis of both tissue and solution cyanide indicated that rice seedlings were quickly able to metabolize exogenous KCN with minor accumulation in plant tissues. In conclusion, significant up-regulation of CYS-A1 suggested that the endogenous pool of cysteine catalyzed by cysteine synthase does not restrict the conversion of exogenous KCN into cyanoalanine through the β-cyanoalanine pathway. However, insufficient responses of the transcription level of NIT4 suggested that NIT enzyme may be a limiting factor for cyanoalanine assimilation by rice seedlings.

Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2 , were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3 , and ferredoxin-encoding gene, pcaA4 , and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation. IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation., (Copyright © 2017 American Society for Microbiology.)

Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that β-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the β-CAS pathway.

Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

Elsholtzia splendens (Lamiaceae) is a copper-tolerant plant species growing on copper deposits in the south of China. Chromatographic separation of n-BuOH extracts from the flowering aerial biomass afforded apigenin-7-O-β-D-glycoside, using macroporous resin, Sephadex™ LH-20 gel, polyamide resin as well as preparative high-performance liquid chromatography (P-HPLC) columns. Chemical structure was elucidated using HPLC/ESI-MS (electrospray ionization-mass spectrometry), Fourier transform infrared (FTIR), and (1)D- and (2)D-nuclear magnetic resonance (NMR). Apigenin-7-O-β-D-glycoside could be the post-harvesting product from E. splendens biomass.

Light-induced brown film (BF) formation by the vegetative mycelium of Lentinula edodes is important for ensuring the quantity and quality of this edible mushroom. Nevertheless, the molecular mechanism underlying this phenotype is still unclear. In this study, a comparative proteomic analysis of mycelial BF formation in L. edodes was performed. Seventy-three protein spots with at least a twofold difference in abundance on two-dimensional electrophoresis (2DE) maps were observed, and 52 of them were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF/MS). These proteins were classified into the following functional categories: small molecule metabolic processes (39%), response to oxidative stress (5%), and organic substance catabolic processes (5%), followed by oxidation-reduction processes (3%), single-organism catabolic processes (3%), positive regulation of protein complex assembly (3%), and protein metabolic processes (3%). Interestingly, four of the proteins that were upregulated in response to light exposure were nucleoside diphosphate kinases. To our knowledge, this is the first proteomic analysis of the mechanism of BF formation in L. edodes . Our data will provide a foundation for future detailed investigations of the proteins linked to BF formation.

Objective: To study clinical efficacy of smoothing Gan reinforcing Shen (SGRS) method in treating poor response of diminished ovarian reserve (DOR) patients in in vitro fertilization and embryo, transfer (IVF-ET)., Methods: Totally 84 DOR patients undergoing IVF-ET were assigned to the experimental group (SGRS Chinese herbs as adjuvant therapy) and the control group according to random digit table, 42 in each group. Patients in the control group received controlled ovarian hyperstimulation (COH) and IVF-ET. Those in the experimental group additionally received basic formula of SGRS method, one dose per day. The dose and use time of recombinant follicle-stimulating hormone (r-FSH) were recorded during ovarian stimulation process. On the injection day of human chorionic gonadotropin (HCG) and serum levels of estradiol (E2) on the oocyte retrieval day were determined using chemiluminescent method. E2 contents in the follicular fluid on the oocyte retrieval day were detected using ELISA. The total number of retrieved oocytes, the number of mature oocytes in metaphase II (M II), the number of normal fertilization [with two pronucleus (2PN)], the number of portable embryos, and the number of good quality embryos were recorded. The correlation between Chinese medical adjuvant therapy and the aforesaid indices were observed. The clinical pregnancy rate and the abortion rate were finally compared between the two groups., Results: The total dose of r-FSH, the E2 level on HCG injection day, the serum E2 level on the oocyte retrieval day, the number of retrieved oocyte, the number of oocytes in M II the number of oocytes with 2PN, the number of portable embryos, and the number of good quality embryos were all positively correlated with Chinese medical adjuvant therapy (P < 0.05, P < 0.01). Compared with the control group, serum E2 levels on the HCG injection day and the oocyte retrieval day obviously increased, the number of retrieved oocytes, the number of oocytes in M II, and the number of portable embryos were increased more in the experimental group with statistical difference (P < 0.05, P < 0.01). There was no statistical significance in the clinical pregnancy rate or the abortion rate between the two groups (P > 0.05)., Conclusion: SGRS Chinese herbs as adjuvant therapy could improve ovarian responsiveness of DOR patients undergoing IVF-ET, increase the number of retrieved oocytes, elevate the quality of oocytes and the number of embryos.

Hydroponic experiments were conducted with rice seedlings (Oryza sativa L. cv. XZX45) exposed to gallium nitrate (Ga(3+)) to investigate the accumulation of Ga in plant tissues and phytotoxic responses. Results showed that phyto-transport of Ga was apparent, and roots were the dominant site for Ga accumulation. The total accumulation rates of Ga responded biphasically to Ga treatments by showing increases at low (1.06-8.52 mg Ga/L) and constants at high (8.52-15.63 mg Ga/L) concentrations, suggesting that accumulation kinetics of Ga followed a typical saturation curve. Higher amount of Ga accumulation in plant tissues led to significant inhibition in relative growth rate and water use efficiency in a dose-dependent manner. DNA-protein cross-links (DPCs) analysis revealed that overaccumulation of Ga in plant tissues positively stimulated formation of DPCs in roots. Likewise, the measure of root cell viability evaluated by Evan blue uptake showed a similar trend. These results suggested that Ga can be absorbed, transported, and accumulated in plant materials of rice seedlings. Overaccumulation of Ga in plant tissues provoked the formation of DPCs in roots, which resulted in cell death and growth inhibition of rice seedlings.

Infertility is a disease of the reproductive system that affects millions of people globally. Reproductive failure is a major medical issue adversely affecting human health in the 21st century. Many factors contribute to infertility, including immune conditions which may lead to immune infertility (immunologic infertility). It is known that specific T helper cells (Th) and their cytokines are involved in the stages of infertility. The aim of this study is to provide a new diagnostic approach to immunologic infertility by investigating the correlation of follicular helper T cells (Tfh) and their secreted cytokines with the autoantibodies in peripheral blood samples from immunologically infertile patients. Thirty (30) patients suffering from immune infertility and 20 control subjects were selected as the sample base for this study. The levels of Tfh, 20 cytokines and 4 antibodies were evaluated for this investigation and evaluated using flow cytometry, antibody chip and ELISA technologies. It was found that, in immunologically infertile patients, Tfh cell numbers were significantly higher than those in the control group. Likewise, seven (7) serum cytokines were expressed to a greater degree in infertile patients compared to the control group. Finally, four (4) antibodies were found to be higher in immunologically infertile patients. The results show that, among patients with immunologic infertility, the levels of Tfh cells and IL-21 were increased significantly in peripheral blood samples and correlate positively with the autoantibodies. IL-12 was positively correlated with the two antibodies, while TNFα was negatively correlated with two additional antibodies. The detection and quantification of Tfh cells, IL-21, IL-12 and TNFα may provide new diagnostic indicators to screen for immunologic infertility.

Hydroponic experiments were conducted with rice seedlings (Oryza sativa L. cv. ND1) exposed to methylene blue (MB) to investigate the accumulation of hydrogen peroxide (H2O2) and activities and gene expression of antioxidant enzymes. Results showed that a linear decrease in relative growth rate and water use efficiency was observed with rice seedlings with increasing MB concentrations. MB-induced accumulation of H2O2 was evident in both roots and shoots. The activities of antioxidative enzymes were analyzed and found to be different at different MB treatment concentrations. The activities of enzymes related to the ascorbate-glutathione cycle were more sensitive to MB treatments than other antioxidative enzymes. Transcript level, by real-time quantitative PCR, of antioxidative enzymes showed that the analyzed genes were differently expressed during different MB concentrations in both roots and shoots. The isoform analysis of superoxide dismutase (SOD) gene showed that the expressions of Cu/ZnSOD and MnSOD were relatively constant, where significant upregulation of FeSOD was observed with rice seedlings exposed to high MB concentrations. Furthermore, the expression of CAT, POD, and MDHAR genes responded biphasically to MB treatments by showing negligible changes at 1.56-15.63 μM MB and significant induction at 31.36-62.52 μM MB. The expression of GR, APX, and DHAR genes showed a remarkable induction to MB. Our results suggest that on transcription level, and in accordance with enzymatic responses, enzymes of GR, APX, and DHAR play central role in the H2O2 detoxification in rice seedlings under MB exposure.

Adsorption behaviors of chromate(VI) on two different iron oxides were investigated through batch experiments under different operational conditions. The operational variables studied included sorbent doses, sorbent species, solution pH, contact time, and initial metal ion concentrations. Both ferri hydroxide and ferri oxohydroxides were capable of removing Cr(VI) from aqueous solution effectively. The extent of metal ion removed from aqueous solution by both sorbents increased with increasing contact time and initial metal ion concentrations, while decreased with elevating solution pH initially. Sorption equilibrium time was observed in 150 min. Equilibrium isotherm data were analyzed using the Langmuir and Freundlich isotherms. The Freundlich model yields a better fit than the Langmuir model. Although kinetic measurements with both sorbents had a similar pattern, ferri hydroxide always showed higher sorptive potential for Cr(VI) than ferri oxohydroxides. Results also indicated that sorptive removal of Cr(VI) by both sorbents was observed to be negative in response to solution pH, due to changes in speciation of Cr(VI) in aqueous solution. At pH lower than 3.0, both iron oxides exhibited much greater capacities of removing Cr(VI) from aqueous solution than other treatments. Therefore, it is to conclude that adsorptive removal of Cr(VI) by both iron oxides is effective. Adsorption capacity is mainly determined by speciation of Cr(VI) in solutions, which is controlled and driven by solution pH.

Solution culture was carried to investigate capacity of synthetic aminopolycarboxylic acids (ethylenediamine tetraacetate, N-hydroxyethylenediaminetriacetic acid, and diethylenetriamine-pentaacetate) for enhancing botanical removal and transport of heavy metals (Cu and Zn) by plants. Biodegradable organic acids (citric acid, malic acid, and oxalic acid) were also selected as alternatives to compare them with synthesized chelating agents for effectiveness. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in nutrient solutions treated with single or combined metal solutions in presence or absence of chelating compounds. Calculation by chemical equilibrium program VISUAL MINTEQ showed that different chelating compounds had various complex potential with Cu(2+) and Zn(2+) ions, in which synthetic chelators exhibited higher complexed capability than biodegradable organic acids. All applied synthetic aminopolycarboxylic acids significantly decreased removal of metal from nutrient solution (p < 0.01), while more or less effects of organic acids supplied on biosorptive potential were observed with all treatments (p > 0.05), compared with the treatment without metal ligands. Synthetic aminopolycarboxylic acids significantly decreased metal concentrations in plant materials in all treatments (p < 0.01). However, biodegradable organic acids decreased metal concentrations in roots (p < 0.01), but enhanced them in shoots (p < 0.01). Results obtained indicated that synthetic aminopolycarboxylic acids decreased uptake of metals by rice seedlings, but translocation of metals complexed within plant materials was evident. Although exogenous biodegradable organic acids showed negligible effect on botanical removal of metals, metals complexed with organic acids was more mobile than those complexed with other chelating agents. These information collected here had important implication for the use of biodegradable metal chelators in transport of essential micronutrients in plant nutrition.

Revegetation and ecological restoration of a Mn mineland are important concerns in southern China. To determine the major constraints for revegetation and select suitable plants for phytorestoration, pedological and botanical characteristics of a Mn mine in Guangxi, southern China were investigated. All the soils were characterized by low pH and low nitrogen and phosphorus levels except for the control soil, suggesting that soil acidity and poor nutrition were disadvantageous to plant growth. In general, the studied mine soils had normal organic matter (OM) and cation exchange capacity (CEC). However, OM (8.9 g/kg) and CEC (7.15 cmol/kg) were very low in the soils from tailing dumps. The sandy texture and nutrient deficiency made it difficult to establish vegetation on tailing dumps. Mn and Cd concentrations in all soils and Cr and Zn concentrations in three soils exceeded the pollution threshold. Soil Mn and Cd were above phytotoxic levels, indicating that they were considered to be the major constraints for phytorestoration. A botanical survey of the mineland showed that 13 plant species grew on the mineland without obvious toxicity symptoms. High Mn and Cd concentrations have been found in the aerial parts of Polygonum pubescens, Celosia argentea, Camellia oleifera, and Solanum nigrum, which would be interesting for soil phytoremediation. Miscanthus floridulus, Erigeron acer, Eleusina indica, and Kummerowia striata showed high resistance to the heavy metal and harsh condition of the soils. These species could be well suited to restore local degraded land in a phytostabilization strategy.

Kinetics of anaerobic ammonium oxidation (anammox) reaction in marine anammox bacterial culture was first investigated. The nitrogen removal performance of the reactor was compared with prediction of Monod model, modified Stover-Kincannon model, first-order and the Grau second-order substrate removal models. Based on calculations, Monod model, modified Stover-Kincannon model and the Grau second-order model proved to be more appropriate to describe the nitrogen removal kinetics of the reactor than first-order model with high determination coefficients of 0.993, 0.993 and 0.991, respectively. According to the modified Stover-Kincannon model, the maximal substrate removal rate (rm) and saturation rate constant (KB) were suggested as 7.37 and 6.41 g N/L/d, respectively. In addition, in light of the Monod model, the saturation concentration (Ks) and the maximal specific substrate removal rate constant (Rm) were determined to be 0.107 g/L and 0.952 g N/g MLVSS/d, respectively. Moreover, model evaluation was carried out by assessing the linear correlation between measured and predicted values. Both kinetics study and model evaluation showed that Monod model, modified Stover-Kincannon model and the Grau second-order substrate removal models could be used to describe the kinetic behavior or design of the marine anammox reactor., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

As a low-cost treatment technology for effluent, the constructed wetlands can be applied to remove the heavy metals from wastewater. Leersia hexandra Swartz is a metal-accumulating hygrophyte with great potential to remove heavy metal from water. In this study, two pilot-scale constructed wetlands planted with L. hexandra (CWL) were set up in greenhouse to treat electroplating wastewater containing Cr, Cu and Ni. The treatment performance of CWL under different hydraulic loading rates (HLR) and initial metal concentrations were also evaluated. The results showed that CWL significantly reduced the concentrations of Cr, Cu and Ni in wastewater by 84.4%, 97.1% and 94.3%, respectively. High HLR decreased the removal efficiencies of Cr, Cu and Ni; however, the heavy metal concentrations in effluent met Emission Standard of Pollutants for Electroplating in China (ESPE) at HLR less than 0.3 m3/m2 d. For the influent of 5 mg/L Cr, 10 mg/L Cu and 8 mg/L Ni, effluent concentrations were below maximum allowable concentrations in ESPE, indicating that the removal of Cr, Cu and Ni by CWL was feasible at considerably high influent metal concentrations. Mass balance showed that the primary sink for the retention of contaminants within the constructed wetland system was the sediment, which accounted for 59.5%, 83.5%, and 73.9% of the Cr, Cu and Ni, respectively. The data from the pilot wetlands support the view that CWL could be used to successfully remove Cr, Cu and Ni from electroplating wastewater.

Responses of free amino acids to botanical assimilation of free cyanide were investigated. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in nutrient solution amended with free cyanide (KCN). Cyanide was analyzed in solution as well as in plant materials to estimate the phyto-assimilation potential. Free amino acids in different parts of plants were also measured to determine metabolic responses to KCN exposure. Phyto-assimilation of KCN was obvious, and the rates were positively correlated to the concentration supplied. Although changes in total amino acid content in plant materials were negligible during KCN metabolism (p > 0.05), responses of different amino acids to KCN treatments were quite different. All treatments with KCN increased the content of proline (Pro) and isoleucine (Ile) in roots significantly compared with control (p < 0.05), while changes of aspartic acid, lysine, and histidine in roots were more evident at higher KCN treatments (p < 0.05). Results indicate that the content of Pro, Ile, and tyrosine showed pronounced increase in shoots of rice seedlings exposed to KCN at 1.44 mg CN/L or higher (p < 0.05). Other amino acids slightly changed in all plant materials exposed to KCN (p > 0.05). Results indicate that specific amino acids in rice seedlings showed positive response to non-toxic concentrations of exogenous KCN. These findings could provide additional insights into the inducible mechanisms underlying the involvement of amino acids in KCN metabolism.