Genome-wide study of Cerrena unicolor 87613 laccase gene family and their mode prediction in association with substrate oxidation.

BACKGROUND: Laccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1). RESULTS: In this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates. CONCLUSIONS: Our findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Bioinformatic Analysis Reveals the Distinct Role of 5'UTR-Specific m6A RNA Modification in Mice Developing Cerebral Cortices.

N6-methyladenosine (m6A) abundantly exists in the cerebral cortex and is emerging as an essential factor in cortical development and function. As the m6A-binding site appears to be dynamically methylated in different RNA regions at the temporal-specific developing stage, it is of value to distinguish the unique character of region- and temporal-specific m6A. Herein, we analyzed the status of temporal-specific m6A within RNA 5' untranslated region (5'UTR) using m6A-methylated sequencing data and transcriptomic sequencing data from 12.5- to 13-day embryonic cerebral cortices and 14-day postnatal ones. We identified sorts of RNAs that are uniquely m6A-methylated in the 5'UTR and sorted them into specific neurological processes. Compared with 3'UTR-m6A-methylated RNAs, 5'UTR-m6A-methylated RNAs showed unique functions and mechanisms in regulating cortical development, especially through the pathway of mRNA transport and surveillance. Moreover, the 5'UTR-specific m6A was associated with neurological disorders as well. The FoxO signaling pathway was then focused by these pathogenic 5'UTR-m6A-methylated RNAs and explored to be involved in the determination of neurological disorders. Additionally, the 5'UTR-m6A modification patterns and transcriptional patterns play independent but cohesive roles in the developing cortices. Our study emphasizes the importance of 5'UTR-specific m6A in the developing cortex and provides an informative reference for future studies of 5'UTR-specific m6A in normal cortical development and neurological disorders.

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.

Hepatic Artery Injection of 131I-Metuximab Combined with Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Prospective Nonrandomized, Multicenter Clinical Trial.

This prospective nonrandomized, multicenter clinical trial was performed to investigate the efficacy and safety of 131I-labeled metuximab in adjuvant treatment of unresectable hepatocellular carcinoma. Methods: Patients were assigned to treatment with transcatheter arterial chemoembolization (TACE) combined with 131I-metuximab or TACE alone. The primary outcome was overall tumor recurrence. The secondary outcomes were safety and overall survival. Results: The median time to tumor recurrence was 6 mo in the TACE + 131I-metuximab group (n = 160) and 3 mo in the TACE group (n = 160) (hazard ratio, 0.55; 95% CI, 0.43-0.70; P < 0.001). The median overall survival was 28 mo in the TACE + 131I-metuximab group and 19 mo in the TACE group (hazard ratio, 0.62; 95% CI, 0.47-0.82; P = 0.001). Conclusion: TACE + 131I-metuximab showed a greater antirecurrence benefit, significantly improved the 5-y survival of patients with advanced hepatocellular carcinoma, and was well tolerated by patients.

[Analysis of complications of high intensity focused ultrasound in treatment of uterine leiomyoma].

OBJECTIVE: To analysis complications and its associated risk factors of high intensity focused ultrasound (HIFU) in treatment of uterine leiomyoma for selecting rationale indicated patients and reducing complications. METHODS: Medical documents of 171 patients with 231 leiomyomas in total treated by HIFU were studied retrospectively. Common complications were categorized and analyzed, the relationship between risk factors and complications were studied. RESULTS: Common complications in treatment of uterine leiomyomas by HIFU were 71.9% (123/171) of abdominal pain, 17.5% (30/171) of vaginal bloody discharge, 8.2% (14/171) of sacroiliac or buttock pain, 7.6% (13/171) of skin blister, 4.7% (8/171) of leg pain, 2.9% (5/171) of hematuria and 1.8% (3/171) of febrile. By logistic regression analysis, the factor correlated with abdominal pain included diameter of uterine leiomyomas, sonication time and average power (P < 0.05). The factor correlated with sacroiliac or buttock pain was uterine leiomyomas located in posteriors of uterine wall (P < 0.05); the factors correlated with vaginal bloody discharge were sonication time and type of uterine leiomyomas (submucous > intramural > subserous, P < 0.05); the factors correlated with skin blister was sonication time (P < 0.05). There were no statistical relationship between multiple factors and leg pain, hematuria, febrile (P > 0.05). CONCLUSION: The modality of high-power and short-term treatment might reduce complications of HIFU ablation.

[Study on the efficacy and safety of ultrasound ablation in treatment of uterine fibroids].

OBJECTIVE: To investigate the safety and efficacy of ultrasound ablation in treatment of uterine fibroids. METHODS: Ninety-nine patients with 117 leiomyomas in total treated by Haifu JC focused ultrasound tumor therapeutic system were enrolled in prospective and non-randomized clinical trial in First Affiliated Hospital of Chongqing Medical University and Academy of Military Medical Sciences. Ultrasound ablation was performed guided by real-time ultrasonography under conscious sedation for single session. All patients were followed up at 6 months after treatment. On the day of treatment and after 1 month, patients were given by magnetic resonance imaging (MRI) exam to evaluate the effect of fibroids ablation. At 3 and 6 months after treatment, the ratio of ablated area and volume reduction of fibroids more than 50% were evaluated by MRI exam again. The symptoms improvements were evaluated by uterine fibroid symptom (UFS) and complications were analyzed by guideline of society of international radiation (SIR). RESULTS: The average ablated area ratio of the target fibroid was (76 ± 24)%. The average reduction in fibroid volume determined by MRI at 3 and 6 months after treatment was (45 ± 21)% and (59 ± 26)%. Which were significantly decreased than those before treatment (P < 0.05). At 6 months after treatment, 84.6% (99/117) of patients showed more than 50% volume reduction, the rate of improved symptom score was 92% (66/72). All patients could resume normal daily activities at 2 hours after treatment. The adverse reactions of SIR C-D included delayed hospitalization, repeat treatment and increased level of nursing. E-F included permanent sequelae and death. In this study, no adverse reactions of C-F were recorded. Common complications (SIR A-B, only observation or simple management without sequelae) were 35% (35/99). Four cases with adverse reactions B of SIR were found, including 2 cases with skin burning of degree II and 2 cases with febrile, they were administered by symptomatic therapy and changing dressing. The other adverse reaction A of SIR included sorness of buttock, vaginal discharge, dysuria and painful urination, they were only suggested by follow-up. CONCLUSION: It was efficacy and safe that ultrasound ablation as a single strategy were used in treatment of uterine fibroids.

Stimuli-responsive nanoscale drug delivery systems for cancer therapy.

Currently, with the rapid development of nanotechnology, novel drug delivery systems (DDSs) have made rapid progress, in which nanocarriers play an important role in the tumour treatment. In view of the conventional chemotherapeutic drugs with many restrictions such as nonspecific systemic toxicity, short half-life and low concentration in the tumour sites, stimuli-responsive DDSs can deliver anti-tumour drugs targeting to the specific sites of tumours. Owing to precise stimuli response, stimuli-responsive DDSs can control drug release, so as to improve the curative effects, reduce the damage of normal tissues and organs, and decrease the side effects of traditional anticancer drugs. At present, according to the physicochemical properties and structures of nanomaterials, they can be divided into three categories: (1) endogenous stimuli-responsive materials, including pH, enzyme and redox responsive materials; (2) exogenous stimuli-responsive materials, such as temperature, light, ultrasound and magnetic field responsive materials; (3) multi-stimuli responsive materials. This review mainly focuses on the researches and developments of these novel stimuli-responsive DDSs based on above-mentioned nanomaterials and their clinical applications.

Expert consensus on image-guided radiofrequency ablation of pulmonary tumors: 2018 edition.

Lung cancer ranks first in incidence and mortality in China. Surgery is the primary method to cure cancer, but only 20-30% of patients are eligible for curative resection. In recent years, in addition to surgery, other local therapies have been developed for patients with numerous localized primary and metastatic pulmonary tumors, including stereotactic body radiation therapy and thermal ablative therapies through percutaneously inserted applicators. Percutaneous thermal ablation of pulmonary tumors is minimally invasive, conformal, repeatable, feasible, cheap, has a shorter recovery time, and offers reduced morbidity and mortality. Radiofrequency ablation (RFA), the most commonly used thermal ablation technique, has a reported 80-90% rate of complete ablation, with the best results obtained in tumors < 3 cm in diameter. Because the clinical efficacy of RFA of pulmonary tumors has not yet been determined, this clinical guideline describes the techniques used in the treatment of localized primary and metastatic pulmonary tumors in nonsurgical candidates, including mechanism of action, devices, indications, techniques, potential complications, clinical outcomes, post-ablation surveillance, and use in combination with other therapies. In the future, the role of RFA in the treatment of localized pulmonary tumors should ultimately be determined by evidence from prospective randomized controlled trials comparing sublobar resection or stereotactic body radiation therapy.

[Safety and efficacy of gefitinib for treatment of advanced non-small cell lung cancer].

OBJECTIVE: To evaluate the safety and efficacy of gefitinib as second-line or even third-line treatment for previously treated patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). METHODS: 156 patients with locally advanced NSCLC which were about to undergo progression after previous chemotherapy were eligible for this study. The regimen was oral intake of gefitinib 250 mg once daily in the morning or afternoon until the disease progression or toxicity has become intolerable. The drug was provided by AstroZeneca Company by its Expanded Access Program. RESULTS: 154 such patients were evaluable for response and toxicity assessment. The overall rate of objective response and disease control was 28.6% (44/154) and 89.6% (138/154). The median duration of response was 7. 5 months. The median time to disease progression (TTP) was 5. 1 months and the median overall survival time (OS) 10.0 months. The actuarial 1-year survival was 41. 0%. The response rate in adenocarcinoma was significantly higher than that in squamous carcinoma (P = 0. 026). The risk of disease progression in patients with squamous carcinoma was 1. 7 times as much as that of adenocarcinoma patients ( P = 0. 011) , and the risk of death in male was 2. 0 times as much as that in female ( P = 0. 002). At least one of these adverse events would be observed in 40.9% (63/154) of these patients, which, however was mild and reversible. Conclusion Gefitinib is effective and safe as a second-line or third-line treatment for previously treated patients with locally advanced or metastatic non-small cell lung cancer.

[Experimental study of effect of epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in combination with irinotecan].

OBJECTIVE: To assess the optimal regimen and its mechanism of ZD1839 in combination with SN38, the active metabolite of irinotecan (CPT-11), in the colon cancer cell lines HT-29 and LoVo. METHODS: Chou and Talalay method was used to analyze the combination effects of sequencing of ZD1839 and SN38. Western blotting and immunoprecipitation were used to determine the effects of ZD1839 and/or SN38 on their targeted enzymes and downstream markers. Apoptosis was assayed by analyzing histone-associated DNA fragment. RESULTS: Sequential SN38 followed by ZD1839 produced a synergistic effect. In contrast, SN38 following ZD1839 exhibited an antagonist effect. SN38 markedly inhibited topoisomerase I (Topo-I) activity. ZD1839 did not alter epidermal growth factor receptor (EGFR) expression, but resulted in a complete inhibition of EGFR phosphorylation. Sequential ZD1839 followed by SN38 did not show any enhanced inhibition effect on Topo-I activity, phosphorylation of EGFR and one of its downstream markers MAPK. However, simultaneous SN38 plus ZD1839, and sequential SN38 followed by ZD1839 administrations showed modest inhibition effect on EGFR's another downstream marker AKT. The combination schedules also showed prominent influence on cell cycle distribution. ZD1839 maintained SN38-induced DNA damage and apoptosis. CONCLUSION: Sequential SN38 followed by ZD1839 may be a favorable combination schedule.

Oral immunization of mice with vaccine of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit.

OBJECTIVE: To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection. METHODS: Using genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected. RESULTS: The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. CONCLUSION: The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Chinese expert consensus workshop report: Guidelines for thermal ablation of primary and metastatic lung tumors.

Although surgical resection is the primary means of curing both primary and metastatic lung cancers, about 80% of lung cancers cannot be removed by surgery. As most patients with unresectable lung cancer receive only limited benefits from traditional radiotherapy and chemotherapy, many new local treatment methods have emerged, including local ablation therapy. The Minimally Invasive and Comprehensive Treatment of Lung Cancer Branch, Professional Committee of Minimally Invasive Treatment of Cancer of the Chinese Anti-Cancer Association has organized multidisciplinary experts to develop guidelines for this treatment modality. These guidelines aim at standardizing thermal ablation procedures and criteria for selecting treatment candidates and assessing outcomes; and for preventing and managing post-ablation complications.

[Clinical analysis and prevention of complications associated with ultrasound-guided percutaneous thick needle biopsy].

OBJECTIVE: To investigate the occurrence of complications in US-guided percutaneous biopsy using core (>19G) gauge cutting needle. METHODS: A retrospective analysis of 5366 US-guided thick needle biopsies was conducted to analyze the incidence of complications after biopsy at different positions. RESULTS: The total incidence of complications was 1.08%, including most frequent hemorrhage, pneumothorax, hemotysis and infection. CONCLUSION: Ultrasound-guided automatic biopsy is easy to operate and safe, and strict execution of the procedures can lower the incidence of the complications.