Ancestral sequence reconstruction of the prokaryotic three-domain laccases for efficiently degrading polyethylene.

Biodegradation of polyethylene (PE) plastics is environmentally friendly. To obtain the laccases that can efficiently degrade PE plastics, we generated 9 ancestral laccases from 23 bacterial three-domain laccases through ancestral sequence reconstruction. The optimal temperatures of the ancestral laccases were between 60 °C-80 °C, while their optimal pHs were at 3.0 or 4.0. Without substrate pretreatment and mediator addition, all the ancestral laccases can degrade low-density polyethylene (LDPE) films at pH 7.0 and 60 °C. Among them, Anc52, which shared low sequence identity (18 %-41.7 %) with the reported PE-degrading laccases, was the most effective for LDPE degradation. After the catalytic reactions at 90 °C for 14 h, Anc52 (0.2 mg/mL) induced clear wrinkles and deep pits on the PE film surface detected by scanning electron microscope, and its carbonyl and hydroxyl indices reached 2.08 and 2.42, respectively. Then, we identified the residues 203 and 288 critical for PE degradation through site-directed mutation on Anc52. Moreover, Anc52 be activated by heat treatment (60 °C and 90 °C) at pH 7.0, which gave it a high catalytic efficiency (kcat/Km= 191.73 mM-1·s-1) and thermal stability (half-life at 70 °C = 13.70 h). The ancestral laccases obtained here could be good candidates for PE biodegradation.

Efficient Production of a Thermostable Mutant of Transglutaminase by Streptomyces mobaraensis.

The transglutaminase (TGase) from Streptomyces mobaraensis is widely used to improve the texture of protein-based foods. However, wild-type TGase is not heat-resistant, which is unfavorable for its application. In this study, we successfully constructed a S. mobaraensis strain that can efficiently produce TGm2, a thermostable mutant of S. mobaraensis TGase. First, S. mobaraensis DSM40587 was subjected to atmospheric room temperature plasma mutagenesis, generating mutant smY2022 with a 12.2-fold increase in TGase activity. Then, based on the double-crossover recombination, we replaced the coding sequence of the TGase with that of TGm2 in smY2022, obtaining the strain smY2022-TGm2. The extracellular TGase activity of smY2022-TGm2 reached 61.7 U/mL, 147% higher than that of smY2022. Finally, the catalytic properties of TGm2 were characterized. The half-life time at 60 °C and specific activity of TGm2 reached 64 min and 71.15 U/mg, 35.6- and 2.9-fold higher than those of the wild-type TGase, respectively. As indicated by SDS-PAGE analysis, TGm2 exhibited demonstrably better protein cross-linking ability than the wild-type TGase at 70 °C, although both enzymes shared a similar ability at 40 °C. With improved enzyme production and thermal stability, smY2022-TGm2 could be a competitive strain for the industrial production of transglutaminase.

Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions.

Streptomyces mobaraenesis transglutaminase can catalyze the cross-linking of proteins, which has been widely used in food processing. In this study, we rationally modified flexible regions to further improve the thermostability of FRAPD-TGm2 (S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L), a stable mutant of the transglutaminase constructed in our previous study. First, five flexible regions of FRAPD-TGm2 were identified by molecular dynamics simulations at 330 and 360 K. Second, a script based on Rosetta Cartesian_ddg was developed for virtual saturation mutagenesis within the flexible regions far from the substrate binding pocket, generating the top 18 mutants with remarkable decreases in folding free energy. Third, from the top 18 mutants, we identified two mutants (S116A and S179L) with increased thermostability and activity. Finally, the above favorable mutations were combined to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), exhibiting a half-life of 132.38 min at 60 °C (t1/2(60 °C)) and a specific activity of 79.15 U/mg, 84 and 21% higher than those of FRAPD-TGm2, respectively. Therefore, the current result may benefit the application of S. mobaraenesis transglutaminase at high temperatures.

Prognostic value of CD247 in patients with head and neck squamous cell carcinoma: bioinformatic analysis of TCGA database.

Background: Head and neck squamous cell carcinoma (HNSC) is the 7th most common type of cancer in the world. Through the advantages of The Cancer Genome Atlas (TCGA) large-scale sequencing-based genome analysis technology, we can explore the potential molecular mechanisms that can improve the prognosis of HNSC patients. Methods: The HNSC transcriptome and clinical data were downloaded from TCGA database. A univariate survival analysis and differential expression analysis were conducted to obtain the intersection gene set. A protein-protein interaction (PPI) analysis, modular analysis, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were then conducted to identify the hub genes. Clinical correlation analysis, univariate and multivariate Cox regression analyses were performed on the identified hub genes to determine the prognostic impact of hub genes on HNSC patients. Results: In total, 601 intersecting gene sets were obtained. A modular analysis was conducted, and the highest scoring module was 19.304. Based on the GO/KEGG enrichment analysis results, CD247 molecule (CD247) was ultimately selected as the gene for this study. The CD247 were divided into a high-expression group and a low-expression group. The Kaplan-Meier survival curve analysis showed that there was a significant difference between the 2 groups (P<0.0001). The median survival time of the low-expression CD247 group was 30.9 months, and the 5-year survival rate was 36.4%. While the median survival time of the high-expression CD247 group was 68.8 months, and the 5-year survival rate was 52.3%. The clinical correlation analysis showed that CD247 was significantly negatively correlated with pathological tumor stage (pT) and pathological nodal extracapsular spread. Gene Set Enrichment Analysis (GSEA) showed that CD247 activating KEGG pathway hsa04650 and hsa04660. Conclusions: CD247 is an independent protective factor in the prognosis of HNSC patients. By activating the hsa04650 and hsa04660 pathways, the expression of interferon gamma, interleukin (IL)-2, and IL-10 is promoted, which in turn improves the tumor immune monitoring ability of the body, induces tumor cell apoptosis, and inhibits tumor cell growth. CD247 is a potential target for improving the clinical treatment effect of HNSC and the prognosis of patients.

Prognostic impact of pretreatment serum superoxide dismutase activity in patients with locoregionally advanced nasopharyngeal carcinoma.

PURPOSE: To evaluate the prognostic effect of pretreatment serum superoxide dismutase (SOD) activity in locoregionally advanced nasopharyngeal carcinoma. METHODS: A total of 498 patients diagnosed with stage III-IVA nasopharyngeal carcinoma between January 2013 and December 2016 were involved in this study. The X-tile program was used to determine the cut-off value of pretreatment serum SOD activity based on disease-free survival. Kaplan-Meier methods and Cox proportional hazards models were used to evaluate the impact of serum SOD levels on survival outcomes. The receiver operating characteristic (ROC) curve analysis was used to compare the prognostic value of clinical stage, pretreatment serum SOD level, and the combination of them regarding disease-free survival. RESULTS: Based on the X-tile plot, the optimal cutoff value of pretreatment serum SOD activity for disease-free survival was 146.0U/mL. As a dichotomous variable, SOD was significantly higher in non-keratinizing differentiated disease (P = 0.027) and early T stage (P = 0.011). Compared with the lower subset, higher SOD activity predicted an inferior 3-year rates of overall survival (84.6 vs. 94.7%, P < 0.001), distant metastasis-free survival (78.3 vs. 92.8%, P < 0.001) and disease-free survival (78.2 vs. 92.8%, P < 0.001). Multivariate analysis verified that the SOD activity was an independent prognostic indicator to predict distant metastasis, disease progression, and death. The area under the ROC curve (AUC) of the combination was superior to that of clinical stage or SOD alone for disease-free survival (both P < 0.01). CONCLUSION: Serological SOD activity before treatment is an important prognostic indicator for patients with stage III-IV non-metastatic nasopharyngeal carcinoma undergoing chemoradiation therapy.

Noncoding RNAs and hyperthermic intraperitoneal chemotherapy in advanced gastric cancer.

Gastric cancer (GC) is one of the most common malignant tumors globally. About 20-30% of patients with gastric cancer show peritoneal implantation metastasis at the first diagnosis. Peritoneal metastasis is responsible for 70% of deaths of patients with advanced gastric cancer. Although there are many ways to treat advanced gastric cancer, the prognosis of patients with recurrence is unsatisfactory. An auxiliary treatment with hyperthermic intraperitoneal chemotherapy (HIPEC), is an internationally recognized recommended treatment for advanced gastric cancer. A series of clinical trials have shown that HIPEC significantly improves the overall survival of patients with cancer. Compared with the cytoreductive surgery (CRS) alone, HIPEC combined with CRS markedly reduced the rate of peritoneal metastasis in patients with ovarian cancer and colorectal cancer. It has been demonstrated that HIPEC alters transcription of many genes by affecting non-coding RNAs, which may contribute to the suppressive effect of HIPEC on the synthesis of nucleic acids and proteins in cancer cells. This paper reviews the recent advances in understanding the role of non-coding RNAs in tumor invasion and metastasis of advanced gastric cancer. We also consider changes in noncoding RNA levels and other molecules in advanced gastric cancer cases treated with HIPEC. We hope that our review will provide a reference for future research on molecular epidemiology and etiology of advanced gastric cancer and promote precise treatment of this malignancy using HIPEC.

Endocannabinoid System Unlocks the Puzzle of Autism Treatment via Microglia.

Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder and characterized by early childhood-onset impairments in social interaction and communication, restricted and repetitive patterns of behavior or interests. So far there is no effective treatment for ASD, and the pathogenesis of ASD remains unclear. Genetic and epigenetic factors have been considered to be the main cause of ASD. It is known that endocannabinoid and its receptors are widely distributed in the central nervous system, and provide a positive and irreversible change toward a more physiological neurodevelopment. Recently, the endocannabinoid system (ECS) has been found to participate in the regulation of social reward behavior, which has attracted considerable attention from neuroscientists and neurologists. Both animal models and clinical studies have shown that the ECS is a potential target for the treatment of autism, but the mechanism is still unknown. In the brain, microglia express a complete ECS signaling system. Studies also have shown that modulating ECS signaling can regulate the functions of microglia. By comprehensively reviewing previous studies and combining with our recent work, this review addresses the effects of targeting ECS on microglia, and how this can contribute to maintain the positivity of the central nervous system, and thus improve the symptoms of autism. This will provide insights for revealing the mechanism and developing new treatment strategies for autism.

Pan-Cancer Analysis of Genomic and Prognostic Characteristics Associated With Coronavirus Disease 2019 Regulators.

Background: Cancer patients are alleged to have poor coronavirus disease 2019 (COVID-19) outcomes. However, no systematic or comprehensive analyses of the role and mechanisms of COVID-19 receptor-related regulators in cancer are available. Methods: We comprehensively evaluated the genomic alterations and their clinical relevance of six COVID-19 receptor-related regulators [transmembrane serine protease 2 (TMPRSS2), angiotensinogen (AGT), angiotensin-converting enzyme 1 (ACE1), solute carrier family 6 member 19 (SLC6A19), angiotensin-converting enzyme 2 (ACE2), and angiotensin II receptor type 2 (AGTR2)] across a broad spectrum of solid tumors. RNA-seq data, single nucleotide variation data, copy number variation data, methylation data, and miRNA-mRNA interaction network data from The Cancer Genome Atlas (TCGA) of 33 solid tumors were analyzed. We assessed the sensitivities of drugs targeting COVID-19 receptor-related regulators, using information from the Cancer Therapeutics Response Portal database. Results: We found that there are widespread genetic alterations of COVID-19 regulators and that their expression levels were significantly correlated with the activity of cancer hallmark-related pathways. Moreover, COVID-19 receptor-related regulators may be used as prognostic biomarkers. By mining the genomics of drug sensitivities in cancer databases, we discovered a number of potential drugs that may target COVID-19 receptor-related regulators. Conclusion: This study revealed the genomic alterations and clinical characteristics of COVID-19 receptor-related regulators across 33 cancers, which may clarify the potential mechanism between COVID-19 receptor-related regulators and tumorigenesis and provide a novel approach for cancer treatments.

MicroRNA-124-3p inhibited progression of nasopharyngeal carcinoma by interaction with PCDH8 and the inactivation of PI3K/AKT/mTOR pathway.

Nasopharyngeal carcinoma (NPC) is characterised by distinct geographical distribution and is particularly prevalent in Asian countries. But the mechanisms related to the progression of nasopharyngeal carcinoma (NPC) are not completely understood. MiR-124-3p functions as a tumor suppressor in many kinds of human cancers. Here, we explored the effects and mechanism of miR-124-3p on the proliferation and colony formation in NPC. In our study, we reported that miR-124-3p was significantly downregulated in NPC tissues and cell lines. Overexpression miR-124-3p decreased NPC cell proliferation and colony formation abilities. Meanwhile, knockdown miR-124-3p increased proliferation and colony formation abilities. Additionally, dual-luciferase assay showed that miR-124-3p could positively regulated PCDH8 by targeting its 3'-UTR. Overexpression of PCDH8 could partially rescue the proliferation and colony formation role of miR-124-3p inhibitor. Our study indicated that miR-124-3p played a tumor suppressor by directly interacting with PCDH8 and inhibiting the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Overall, we found that miR-124-3p inhibited the activation of the PI3K/AKT/mTOR signaling pathway in NPC by interacting with PCDH8. Thus, PCDH8 may be a potential molecular target that impeded NPC proliferation and colony formation.

Erlotinib: An enhancer of radiation therapy in nasopharyngeal carcinoma.

The aim of this study was to explore the effects of erlotinib combined with radiation on human nasopharyngeal carcinoma (NPC) radiosensitivity using the CNE1 and CNE2 cell lines. Human NPC cells were treated with erlotinib and/or radiation. The effect of erlotinib on the radiosensitivity of the cells was detected using a clonogenic cell survival assay. The rate of apoptosis and the cell cycle were evaluated using flow cytometry. An NPC xenograft model in NOD-SCID mice was used to evaluate the efficacy of the combination therapy of erlotinib with radiation. Erlotinib enhanced the sensitivity of the CNE1 and CNE2 cells to radiation, with sensitization enhancement ratios (SERs) of 1.076 and 1.109, respectively. Erlotinib combined with radiation induced G2/M phase cell cycle arrest in the two cell lines. The mouse tumor model demonstrated a significant reduction in NPC tumor volume in mice treated with erlotinib in combination with radiation when compared with that in mice treated with radiation alone. Erlotinib combined with radiation provoked G2-M phase cell cycle arrest, thereby enhancing the sensitivity of the NPC cells to radiation.