Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Characterisation of a laccase isolated from Trametes hirsuta and its application in the oligomerisation of phenolic compounds.

Phenolic compounds are widely distributed in nature and industrial environment, and their detoxification or bioactive enhancement is of great value to environmental protection and industrial development. Laccases are multicopper oxidases that catalyse the oligo- or polymerisation of phenolic compounds. Identifying new laccase producers and investigating their application potential are of great importance. In this study, a white-rot fungus, Trametes hirsuta EZ1, with significantly high laccase productivity was isolated. The optimum conditions were studied for the maximum fermentation of extracellular laccase, which was achieved at 150 U/mL with a medium containing 10% strain EZ1, 7% maltodextrin, 1.5% peptone, and 0.5 mM Cu2+, and incubation at initial pH 6.0, 32 °C, and 180 rpm for nine days. Subsequently, a 70-kDa laccase was purified that showed activity over a wide range of temperature and pH, sensitivity to many metal ions and sodium dodecyl sulphate, and high tolerance to organic solvents. Purified laccase showed a significant unreported effect by catalysing catechol or ferulic acid into dimers, trimers, and tetramers or caffeic acid into dimers, trimers, tetramers, and pentamers. The oligomeric mixtures exhibited increased antioxidative capacity compared to that of each parent monomer, except for caffeic acid derivatives. Our study offers a novel strain source for laccase production and broadens its application in the enhancement of bioactive compounds.

Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana.

Beauveria bassiana is a critical entomopathogenic fungus for pest biocontrol, whose efficiency depends on fungal development and stress resistance. Unlike its revealed location in plasma membrane patches in other organisms, B. bassiana Sur7 specifically localized in vacuoles. This vacuolar Sur7 was previously demonstrated to affect stress tolerance, hyphal development and virulence. There, however, remain more mechanistic details to be explored. In this study, transcriptomics and metabolomics were applied to investigate the mechanism of vacuolar Sur7. Analyses of transcriptomics and metabolomics displayed many differentially expressed genes and abundant metabolites in response to Sur7 loss, respectively. Together with genes associated with vacuolar biofunction (including transportation and hydrolysis), the altered metabolites contributed to cell wall construction and stress resistance. Particularly, an N-acetylglucosamine-associated Brg1/Nrg1 pathway was enriched and partially affected by Sur7. Absence of Sur7 changed the expression level of Brg1/Nrg1 pathway-related transcript factors, which interfered with downstream phenotype of sporulation. In addition, Sur7 was involved in the accumulation of sphingoid bases, which may affect sphingolipid-related signaling pathway. Although experimental evidence is further required, our studies provide a preliminary framework for future exploring the regulatory mechanism of Sur7, and give a new version of metabolic agency connecting Sur7 and downstream signaling pathway.

Transcriptomic analysis of Sur7-mediated response of Beauveria bassiana to different nutritional conditions.

Integrity of the cell wall is requisite for fungal growth and function. Sur7 governs cell wall composition, and affects conidial sporulation and germination in Beauveria bassiana, a filamentous entomopathogenic fungus. The role of Sur7 in fungal growth on various nutrients remains unclear. We have previously reported that Sur7 deletion results in the attenuation of B. bassiana growth on supplemented Sabouraud dextrose agar (SDAY) and minimal Czapek-Dox agar (CDA) compared to wild type (WT). Here, we used transcriptomic analysis to compare WT and Sur7 mutant (ΔSur7) responses to CDA and SDAY. Growth on CDA, compared with that on SDAY, affected the expression of more genes in the WT than in the mutant. Differentially expressed genes were enriched for transportation process terms in the ΔSur7 mutant and metabolic process terms in the WT. Different processes were repressed in the ΔSur7 (metabolic process) and WT (ribosome synthesis) cells. Despite the shared enrichment of nitrogen metabolism genes, differentially expressed genes were enriched in distinct saccharide-energy metabolism terms in each strain. We conclude that Sur7 ensures the growth of B. bassiana in a minimal medium by influencing the expression of genes involved in the consumption of sucrose via specific energy metabolism pathways.

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of ß-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

Expression and biochemical characterization of a novel chitinase ChiT-7 from the metagenome in the soil of a mangrove tidal flat in China.

Chitinases play an important role in the process of chitin bioavailability. In this study, we cloned a new chitinase gene and characterized its recombinant protein. The new 1251 bp gene of chitinase (ChiT-7) was cloned from the metagenome of the mangrove tidal flat soil in the city of Zhangzhou in Fujian Province (China) by genome walking. The gene encoded a mature protein with 381 amino acids, which manifested certain sequence similarity (59% identity) to characterized GH18 chitinases. The mature protein of ChiT-7 was successfully expressed in E. coli BL21 (DE3). After purification, the specific activity of the recombinant enzyme was 0.63 U/mg at the optimal pH of 6.0 and the optimal temperature of 45 °C. The rChiT-7 was active over a wide pH range, and the residual enzyme activity reached 80% or higher at 30 °C-50 °C. rChiT-7 hydrolyzed colloidal chitin with (GlcNAc)2 and GlcNAc as the main final products. Structural analysis of ChiT-7 indicated that ChiT-7 could be a processive chitinase. rChiT-7 manifested characteristics analogous to those of fungi and actinomycetes and exhibited sequence homology.

A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris.

An agar-degrading bacterium, strain BN3, was isolated from a coastal soil sample collected in Taiwan Strait, China and identified to be a novel species of the genus Microbulbifer. The gene (N3-1) encoding for a novel ß-agarase from the isolate was cloned and sequenced. It encoded a mature protein with 274 amino acids and a calculated molecular mass of 34.3 kDa. The deduced amino acid sequence manifested sequence similarity (61-84% identity) to characterized ß-agarases in the glycoside hydrolase family 16. The recombinant agarase was hyper-produced extracellularly using Pichia pastoris as the host. After induction in a shake flask for 96 h, the yield of recombinant N3-1 protein reached 0.406 mg/mL, and the enzyme activity attained 502.1 U/mL. The enzyme purified by ion exchange chromatography displayed a specific activity of 6447 U/mg at pH 6.0 and 50 °C. The optimal pH and temperature for agarase activity were approximately 6 and 50 °C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type ß-agarase, capable of hydrolyzing agarose and Gracilaria lemaneiformis, with neoagarobiose and neoagarotetraose as the final main products.

Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformis agar: A comparative study.

Hydrolysis of Gracilaria lemaneiformis agar by ß-agarase was compared with HCl hydrolysis. The results showed that optimum catalysis conditions for the ß-agarase were pH 7.0 at 45°C. Mass spectroscopy, thin-layer chromatography and GPC results showed that the polymerization degrees of the hydrolysis products by the ß-agarase were mainly four, six and eight (more specific than the hydrolysate by HCl). The enzymatic degradation products of agar were distinctly different from those of HCl hydrolysis in the ratios among galactose and 3,6-anhydro-galactose and sulfate group contents. The NMR spectrometry proved that the products of ß-agarase were neoagaroligosaccharides, which was not found in the agarolytic products by HCl. The neoagarotetraose inhibited tyrosinase activity competitively with the KI value of 16.0mg/ml. Hydroxyl radical-scavenging ability of neoagaroligosaccharides was much greater than that of agar HCl hydrolysate. This work suggests that neoagaroligosaccharide products produced by our ß-agarase could be more effective in function than products from acid hydrolysis.

Posterior reversible encephalopathy syndrome in patients with hematologic tumor confers worse outcome.

BACKGROUND: This study aimed to evaluate the clinical features of posterior reversible encephalopathy syndrome (PRES) in children. METHODS: The medical records of 31 patients from five medical centers who were diagnosed with PRES from 2001 to 2013 were retrospectively analyzed. In the 31 patients, 16 were males, and 15 females, with a median age of 7 years (3-12 years). Patients younger than 10 years accounted for 74.2% of the 31 patients. RESULTS: Seizure, the most common clinical sign, occurred in 29 of the 31 patients. Visual disturbances were also observed in 20 patients. Cerebral imaging abnormalities were bilateral and predominant in the parietal and occipital white matter. In this series, three patients died in the acute phase of PRES. One patient had resolution of neurologic presentation within one week, but no apparent improvement in radiological abnormalities was observed at eight months. One patient showed gradual recovery of both neurologic presentation and radiological abnormalities during follow-up at eight months. One patient developed long-term cortical blindness. All of the PRES patients with hematologic tumor had a worse prognosis than those without hematologic tumor. CONCLUSIONS: Seizure is a prevalent characteristic of children with PRES. Poor prognosis can be seen in PRES patients with hematologic tumor.

Highly efficient expression and characterization of a ß-mannanase from Bacillus subtilis in Pichia pastoris.

A ß-mannanase gene (Man5) from Bacillus subtilis BS5 was cloned by PCR and integrated into the genome of Pichia pastoris GS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43 kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96 H, the recombinant Man5 protein reached 375 µg/mL in concentration, with an enzyme activity of 892 U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978 U/mg. The optimum temperature and pH of the recombinant Man5 were 50 °C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.

High phylogenetic diversity of glycosyl hydrolase family 10 and 11 xylanases in the sediment of Lake Dabusu in China.

Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

Antifungal and antiviral products of marine organisms.

Marine organisms including bacteria, fungi, algae, sponges, echinoderms, mollusks, and cephalochordates produce a variety of products with antifungal activity including bacterial chitinases, lipopeptides, and lactones; fungal (-)-sclerotiorin and peptaibols, purpurides B and C, berkedrimane B and purpuride; algal gambieric acids A and B, phlorotannins; 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy)phenol, spongistatin 1, eurysterols A and B, nortetillapyrone, bromotyrosine alkaloids, bis-indole alkaloid, ageloxime B and (-)-ageloxime D, haliscosamine, hamigeran G, hippolachnin A from sponges; echinoderm triterpene glycosides and alkene sulfates; molluscan kahalalide F and a 1485-Da peptide with a sequence SRSELIVHQR; and cepalochordate chitotriosidase and a 5026.9-Da antifungal peptide. The antiviral compounds from marine organisms include bacterial polysaccharide and furan-2-yl acetate; fungal macrolide, purpurester A, purpurquinone B, isoindolone derivatives, alterporriol Q, tetrahydroaltersolanol C and asperterrestide A, algal diterpenes, xylogalactofucan, alginic acid, glycolipid sulfoquinovosyldiacylglycerol, sulfated polysaccharide p-KG03, meroditerpenoids, methyl ester derivative of vatomaric acid, lectins, polysaccharides, tannins, cnidarian zoanthoxanthin alkaloids, norditerpenoid and capilloquinol; crustacean antilipopolysaccharide factors, molluscan hemocyanin; echinoderm triterpenoid glycosides; tunicate didemnin B, tamandarins A and B and; tilapia hepcidin 1-5 (TH 1-5), seabream SauMx1, SauMx2, and SauMx3, and orange-spotted grouper ß-defensin. Although the mechanisms of antifungal and antiviral activities of only some of the aforementioned compounds have been elucidated, the possibility to use those known to have distinctly different mechanisms, good bioavailability, and minimal toxicity in combination therapy remains to be investigated. It is also worthwhile to test the marine antimicrobials for possible synergism with existing drugs. The prospects of employing them in clinical practice are promising in view of the wealth of these compounds from marine organisms. The compounds may also be used in agriculture and the food industry.

High-level expression of a sika deer (Cervus nippon) Cu/Zn superoxide dismutase in Pichia pastoris and its characterization.

Production of a sika deer Cu/Zn-SOD was achieved in Pichia pastoris after the reconstituted expression vector pPIC9K was transformed into the strain GS115. By employing Saccharomyces cerevisiae secretion signal peptide (α-factor) under the regulation of the methanol-inducible promoter of the gene of alcohol oxidase 1 (AOX1), sika deer Cu/Zn-SOD with a molecular mass of 16kDa was expressed while recombinant sika deer Cu/Zn-SOD with an activity of 3500U/mL was obtained from a 5L bioreactor. After two successive steps of chromatography on DEAE-650C and Superdex75, recombinant sika deer Cu/Zn-SOD was obtained with 13.8% yield, 14.5-fold purification, and a specific activity of 3447U/mg. Its optimum temperature and optimum pH were 40°C and 7.0, respectively.

Pharmacotherapy approaches to antifungal prophylaxis.

INTRODUCTION: Invasive fungal infection (IFI) is a serious problem due to difficulties in early diagnosis and high mortality. Different approaches are adopted for the treatment and management of IFI, including prophylactic, empiric, preemptive and directed strategies. AREAS COVERED: This paper reviews the type of pharmacotherapy used for antifungal prophylaxis in infants with extremely low birth weights, pediatric patients with cardiac disease, preterm neonates, pediatric oncology patients, adult cancer patients with neutropenia, adult patients with hematologic malignancy, hematopoietic stem-cell transplantation recipients, organ transplant recipients, HIV-infected patients, immunosuppressed patients treated with moderate or high doses of corticosteroids, and patients with invasive fusariosis, candidemia, invasive candidiasis, systemic mycoses and immunocompromised patients. EXPERT OPINION: Azole drugs are the drugs most often used in cost-effective antifungal prophylaxis of patients with conditions such as immunodeficiency and cancer, which render them highly susceptible to IFI. Fluconazole is the most outstanding example. However, there are many azoles with different pharmacological characteristics that the physician can choose from. Echinocandins have favorable characteristics that make them useful for treating Candida infections. Antibodies, or their engineered derivatives directed against cell-wall polysaccharides and glycopeptides, and some protein epitopes of Candida albicans, appear to be a promising novel approach for prophylaxis against Candida infection and deserve further in-depth investigations.

[An epidemiological survey of febrile convulsions among pupils in the Wenzhou region].

OBJECTIVE: To study the prevalence and clinical features of febrile convulsion (FC) among pupils in the Wenzhou region, Zhejiang Province, China. METHODS: Using a random stratified cluster sampling method, 6406 children under 12 years from two primary schools of urban areas and two primary schools of rural areas were surveyed. RESULTS: The prevalence of FC was 3.67% (235/6406). Most children (75.7%) experienced their first onset of FC at 6 months to 3 years of age (median: 16 months). The seizures were generalized (95.3%, 224/235), with a duration of less than 10 minutes (86.4%, 203/235). FC was developed into epilepsy in 13 children (5.5%) who all suffered from complex FC. Relapses were noted in 88 cases (37.4%), among whom 38 patients had only 1 recurrence and 50 patients had 2 or more relapses. EEG was performed in 200 cases, among whom 12(6.0%) showed abnormalities. CONCLUSIONS: The prevalence of FC is 3.67% among pupils in the Wenzhou region. The seizures are generalized, with a short duration. A part of complex FC can be developed into subsequent epilepsy.

[The causes and preventive methods of misdiagnosis on wrist scaphoid bone fracture].

OBJECTIVE: To retrospectively analysis the curative effect of wrist scaphoid bone fracture,and explore the causes and preventive methods of misdiagnosis. METHODS: From September 2007 to September 2010,16 patients with wrist scaphoid bone fractures were treated with plaster cast and cannulated screws fixation. There were 10 males and 6 females,ranging in age from 26 to 44 years with an average of 35 years. Among them, 12 cases manifested swelling pain of radial lateral wrist, tenderness at snuffbox area, wrist pain aggravated when stretching wrist joint, thumb or forefinger; 4 cases manifested no obviously symptoms and limited movement; 9 cases were early diagnosed; 5 cases were treated by plaster cast; 4 cases were treated with cannulated screws fixation; Among 7 cases with misdiagnosis, there were 4 cases without obvious symptoms and they were dealt with activating blood to dissipate swelling and pain process in preliminary stage. Four cases were treated with plaster cast and 3 cases with cannulated screws fixation. RESULTS: All the patients were followed up from 3 months to 39 months (averaged 21 months). Among 16 patients, 9 cases were early diagnosis, 7 cases were misdiagnosis and the rate of misdiagnosis was 43.8%. Seven cases with screws fixation were no wound infection. There was 1 case with occurred chronic pain and declining wrist mobility in both plaster cast and screw group, and both of them were misdiagnosed. According to curative effect rating criteria,these 2 cases were classified into moderate, other 14 cases were excellent. CONCLUSION: Wrist scaphoid bone fracture are easy to misdiagnose, so early diagnosis and treatment is particularly important. The main causes of misdiagnosis are nonspecific symptoms at early stage, combination with other injuries, lack of knowledge and ignorance of the further examination. Therefore, detailed inquiries and particular examination, multi-dimensional radiography and CT scan or MRI scan are the main measures for prevention.

Expression of a secretory ß-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization.

A ß-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant ß-glucosidase was expressed and secreted into the culture medium. The maximum recombinant ß-glucosidase activity achieved was 60 U/ml, and ß-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa ß-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70 °C and pH 5.0.

[Application of endobutton in the treatment of acute acromioclavicular joint dislocation].

OBJECTIVE: To study the clinical efficacy of the endobutton in the treatment of acute acromioclavicular joint dislocation by reconstructing coracoclavicular ligaments. METHODS: From October 2008 to January 2010,12 patients with acute acromioclavicular joint dislocation were immobilized with the endobutton. All the patients had the dislocations of or above type III according to Rockwood classification. Among the patients, 9 patients were male and 3 patients were female, with an average age of 55 years (ranged from 31 to 83 years). Eight patients had injuries in the left, and 4 patients in the right. Four patients had accompanied injuries of rib fractures, 2 patients had brain injuries,and 1 patient had femoral fracture. Seven patients were injured by traffic accident, 4 patients were injured by falling down,and 1 patient was sports injuries. All the patients had pain and tenderness at the shoulder, positive piano sign, and shoulder confined activity. The duration from injury to operation ranged from 2 days to 10 days (averaged 6 days). The therapeutic effects were evaluated by Karlsson criteria based on range of motion of acromioclavicular joint, subjective feeling,and postoperative X-ray. RESULTS: All the patients were followed up, and the duration ranged from 4 months to 19 months (averaged 11 months). The motion of the shoulder joint recovered to normal about 15 to 35 days after operation. There were no displacement, dislocation and redislocation occurred. All the patients got A degree results according to Karlsson criteria. CONCLUSION: Reconstruction of coracoclavicular ligament by using the endobutton to treat acute acromioclavicular dislocation of or above type III is a perfect method with advantage of rigid fixation, micro-injury, and early functional exercise.

Glyceollin, a soybean phytoalexin with medicinal properties.

This review covers the biosynthesis of glyceollin and its biological activities including antiproliferative/antitumor action (toward B16 melanoma cells, LNCaP prostate cancer cells, and BG-1 ovarian cancer cells), anti-estrogenic action (through estrogen receptors α- and ß-), antibacterial action (toward Erwinia carotovora, Escherichia coli, Bradyrhizobium japonicum, Sinorhizobium fredii ), antinematode activity, and antifungal activity (toward Fusarium solani, Phakospora pachyrhizi, Diaporthe phaseolorum, Macrophomina phaseolina, Sclerotina sclerotiorum, Phytophthora sojae, Cercospora sojina, Phialophora gregata, and Rhizoctonia solani). Other activities include insulinotropic action and attenuation of vascular contractions in rat aorta.