Elucidation of the Biosynthesis of Griseoluteic Acid in Streptomyces griseoluteus P510.

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.

The Immunologic Profiles of Kawasaki Disease Triggered by Mycoplasma pneumoniae Infection.

OBJECTIVE: We compared the immunologic characteristics of mycoplasma pneumoniae-triggered Kawasaki disease (MP-KD) with Kawasaki disease (KD) not associated with mycoplasma pneumoniae (MP), with mycoplasma pneumoniae-triggered Henoch-Schönlein purpura (MP-HSP), and with healthy controls. METHODS: Complement levels, cellular and humoral immunity were assessed in KD, in MP-KD, in MP-HSP, and in healthy children. RESULTS: Of 622 children with KD, 74 had MP-KD. Complement C3 and CD4/CD8 ratio were significantly increased in MP-KD compared to KD. C3, C4, and the ratio of CD4/CD8 in the MP-KD group were higher than those in the MP-HSP group. IgA and CD56 were lower in the MP-KD group than the MP-HSP group. CONCLUSIONS: Both C3 and polyclonal CD4+ T lymphocytes may be activated in the patients with MP-KD.

Comparative metabolomics and transcriptomics analyses provide insights into the high-yield mechanism of phenazines biosynthesis in Pseudomonas chlororaphis GP72.

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.

Epidemiology and Clinical Characteristics of Henoch-Schönlein Purpura Associated with Streptococcal Infection in 217 Children in Hubei Province, China.

Objectives The objectives of present study were to analyze the association of the streptococcal infection with childhood Henoch-Schönlein purpura (HSP) in China. Methods: We performed a case-control study over a period of five years to evaluate the epidemiology and clinical characteristics of group A ß-hemolytic streptococcal (GABHS) triggered HSP. Results: 1. The frequency of GABHS-triggered HSP was 15.1%, while that of GABHS infection developing HSP in children was 4.7%. 2.The epidemiological characteristics of HSP with streptococcal infection were similar to those of HSP alone. 3. The GABHS-triggered HSP cases had a significantly higher frequency of renal involvement than the noninfectious group. 4. IgA and IgG were significantly increased in the streptococcal infection group than in the noninfectious group, while the levels of C3 and C4 decreased significantly. Conclusions: GABHS infection is the most frequent agent in HSP children, and may aggravate the immune dysfunction and prolong the course of HSP.

[Induction and Anti-Tumor Function of Tertiary Lymphoid Organs].

OBJECTIVE: To induce the development of tertiary lymphoid organs (TLO) in a mouse model of melanoma and to evaluate TLO's functions in antitumor immunity. METHODS: Lymphotoxin-beta receptor (LTßR) was overexpressed in NIH3T3 cells through the lentivirus system and the overexpression efficiency of LTßR in LTßR-NIH3T3 cells was examined. Western blot and qPCR were used to examine the non-canonical nuclear factor (NF)-κB signaling pathway in NIH3T3 cells overexpressing LTßR. B16-OVA melanoma mouse model was constructed to explore the induction of TLO and anti-tumor functions of TLO in LTßR-NIH3T3 cells. RESULTS: LTßR was overexpressed in NIH3T3 cells through the lentivirus system, and flow cytometry showed that the proportion of GFP + cells reached 99%. The overexpression of LTßR activated the non-canonical NF-κB signaling pathway in NIH3T3 cells. Findings from the mouse tumor model suggest that the injection of LTßR-NIH3T3 cells successfully induced the development of lymphoid tissue around the tumor and enhanced the tumor infiltration of T cells and MHCⅡ + macrophages, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice. CONCLUSION: LTßR-NIH3T3 cells promoted anti-tumor immunity by inducing TLO development, which may provide new perspectives for tumor immunotherapy.

rpeA, a global regulator involved in mupirocin biosynthesis in Pseudomonas fluorescens NCIMB 10586.

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.

Engineering of glycerol utilization in Pseudomonas chlororaphis GP72 for enhancing phenazine-1-carboxylic acid production.

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.

Synthesis of cinnabarinic acid by metabolically engineered Pseudomonas chlororaphis GP72.

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 calB3 ) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB3 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 CalB3 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 Cu2+ , H2 O2 , 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.

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58.

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.

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries.

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane-labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.

Engineering Pseudomonas chlororaphis HT66 for the Biosynthesis of Copolymers Containing 3-Hydroxybutyrate and Medium-Chain-Length 3-Hydroxyalkanoates.

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 ß-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 ß-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.

Development of the Static and Dynamic Gene Expression Regulation Toolkit in Pseudomonas chlororaphis.

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 ß-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 PMOK_RS0128085UTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PMOK_RS0116635UTR 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.

Economical Production of Phenazine-1-carboxylic Acid from Glycerol by Pseudomonas chlororaphis Using Cost-Effective Minimal Medium.

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 Fe2+ and NH4+ 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.

Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.

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 (eSpCas9ppD10A ) 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.

Blood glucose levels and the risk of HPV multiple infections in high-grade squamous intraepithelial lesions: A retrospective cross-sectional study of Chinese patients.

Besides the controversy of the association of high glycemic index and glycemic load with precancerous cervical lesions, only a few studies have examined the impact of fasting blood glucose levels on human papillomavirus (HPV) multiple infections. In the present study, we appraised the relationship between blood glucose levels and multiple HPV infections in a population of HPV-positive women with cervical high-grade squamous intraepithelial lesions (HSIL). The present study was designed as a cross-sectional correlative analysis. A total of 560 participants with a pathologically confirmed HSIL with HPV infection were included from a hospital in China during January 1, 2018, and December 31, 2019. The target variables and the outcome variables were the glucose levels at the baseline and HPV multiplicity, respectively. The odds ratio and 95% confidence intervals were calculated to estimate the risk of multiple infections via logistic regression analysis. The average age of the 560 participants was 44.63 ± 10.61 years; the nonlinear relationship was detected between the glucose levels and multiplicity of HPV, with an inflection point at 5.4. After adjusting for the full range of variables, the effect sizes and confidence intervals for the left and right sides of the inflection points were found to be 0.379 (0.196-0.732) and 5.083 (1.592-16.229), respectively. In this cross-sectional study, both high and low blood glucose levels increased the risk of multiple HPV infections, demonstrating a U-shaped relationship between the blood glucose levels and multiple HPV infections.

Enhanced production of 2-hydroxyphenazine in Pseudomonas chlororaphis GP72.

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 µg ml(-1), compared with just 5 µg ml(-1) for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.

[Laboratory diagnosis of HIV/AIDS patients complicated with Pneumocystis jirovecii pneumonia].

Pneumocystis jirovecii was detected in sputum samples and bronchoalveolar lavage fluid (BALF) obtained from HIV/AIDS patients complicated with Pneumocystis jirovecii pneumonia by Giemsa staining. CD4+ T lymphocytes of 500 patients were counted by flow cytometer. P. jirovecii positive rate in sputum samples (46.8%, 845/1 806) significantly lower than that of BALF (55.8%, 10(6)/190) (P < 0.05). The proportion of patients developing clinical symptoms in P. jirovecii positive cases (96.6%, 816/845) was higher than that of P. jirovecii negative cases (64.0%, 615/961) (P < 0.05). P. jirovecii positive rate increased with the decrease of CD4+ T lymphocyte number. P. jirovecii positive rates in cases with CD4+ > 200 x 10(6)/L, CDC 200 x 10(6)/L-100 x 10(6)n/L, and CD4+ < 100x10(6)/12.0% (6/50), 39.0%( 39/100), 54.6% (191/350), respectively (P < 0.05). Giemsa staining is an efficient, simple and feasible method for P. jirovecii detection, relying on the experience and skill of the operator.

Epidemiology and Clinical Characteristics of Henoch-Schönlein Purpura Associated with Epstein-Barr Virus Infection.

BACKGROUND: Henoch-Schönlein purpura (HSP) is an immune-mediated vasculitis, and the formation of immune complexes may be triggered by exposure to Epstein-Barr virus (EBV) infection. METHODS: We performed a five-year case-control study to evaluate the epidemiology and clinical characteristics of HSP associated with EBV infection. RESULTS: The incidence of EBV-triggered HSP was 4.2%, while EBV infection in children with HSP was 0.9%; The EBV-triggered HSP cases had a significantly higher frequency of abdominal pain than the Mycoplasma Pneumoniae (MP)-triggered HSP group (χ2 = 8.024, p = 0.005); Significant differences were observed in the duration of abdominal pain (Z = -1.935, p = 0.027) between the two groups; C3 (t = 9.709, p < 0.001), IgA (t = 20.39, p < 0.001) and IgG (t = 6.407, p < 0.001) were significantly increased in the EBV infection group than those in the healthy control group. Notably, significantly higher proportion of CD19 (t = 6.773, p < 0.001) and lower proportion of CD56 (t = 11.13, p < 0.001) was found in EBV infection group compared with healthy control group. The IgA level was higher than that of the non-infectious group (t = 2.162, p = 0.032), but their CD4/CD8 ratio (t = 10.070, p < 0.001) and CD56 proportion (t = 2.096, p = 0.037) were significantly lower. CONCLUSIONS: Both cellular and humoral immunity were involved in the pathogenesis of EBV-triggered HSP, leading to increased production of inflammatory mediators and immunoglobulins. Those events may cause or promote the development of systemic vessel vasculitis.

Identification of a Novel Bioactive Phenazine Derivative and Regulation of phoP on Its Production in Streptomyces lomondensis S015.

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.

Biosynthesis and Characterization of Medium-Chain-Length Polyhydroxyalkanoate with an Enriched 3-Hydroxydodecanoate Monomer from a Pseudomonas chlororaphis Cell Factory.

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 ß-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.