Regulation of the Gα-cAMP/PKA signaling pathway in cellulose utilization of Chaetomium globosum.

BACKGROUND: The canonical heterotrimeric G protein-cAMP/PKA pathway regulates numerous cellular processes in filamentous fungi. Chaetomium globosum, a saprophytic fungus, is known for producing many secondary metabolites, including cytotoxic chaetoglobosin A (ChA), as well as abundant cellulase and xylanase. RESULTS: Here we report on the functional characterization of this signaling pathway in C. globosum. We blocked the pathway by knocking down the putative Gα-encoding gene gna1 (in the pG14 mutant). This led to impaired cellulase production and significantly decreased transcription of the major cellulase and xylanase genes. Almost all the glycohydrolase family genes involved in cellulose degradation were downregulated, including the major cellulase genes, cel7a, cel6a, egl1, and egl2. Importantly, the expression of transcription factors was also found to be regulated by gna1, especially Ace1, Clr1/2 and Hap2/3/5 complex. Additionally, carbon metabolic processes including the starch and sucrose metabolism pathway were substantially diminished, as evidenced by RNA-Seq profiling and quantitative reverse transcription (qRT)-PCR. Interestingly, these defects could be restored by simultaneous knockdown of the pkaR gene encoding the regulatory subunit of cAMP-dependent PKA (in the pGP6 mutant) or supplement of the cAMP analog, 8-Br-cAMP. Moreover, the Gα-cAMP/PKA pathway regulating cellulase production is modulated by environmental signals including carbon sources and light, in which VelB/VeA/LaeA complex and ENVOY probably work as downstream effectors. CONCLUSION: These results revealed, for the first time, the positive role of the heterotrimeric Gα-cAMP/PKA pathway in the regulation of cellulase and xylanase utilization in C. globosum.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification.

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

Flexible nano-liposomes-encapsulated recombinant UL8-siRNA (r/si-UL8) based on bioengineering strategy inhibits herpes simplex virus-1 infection.

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection.

Enhanced Photodynamic Therapy Synergizing with Inhibition of Tumor Neutrophil Ferroptosis Boosts Anti-PD-1 Therapy of Gastric Cancer.

For tumor treatment, the ultimate goal in tumor therapy is to eliminate the primary tumor, manage potential metastases, and trigger an antitumor immune response, resulting in the complete clearance of all malignant cells. Tumor microenvironment (TME) refers to the local biological environment of solid tumors and has increasingly become an attractive target for cancer therapy. Neutrophils within TME of gastric cancer (GC) spontaneously undergo ferroptosis, and this process releases oxidized lipids that limit T cell activity. Enhanced photodynamic therapy (PDT) mediated by di-iodinated IR780 (Icy7) significantly increases the production of reactive oxygen species (ROS). Meanwhile, neutrophil ferroptosis can be triggered by increased ROS generation in the TME. In this study, a liposome encapsulating both ferroptosis inhibitor Liproxstatin-1 and modified photosensitizer Icy7, denoted LLI, significantly inhibits tumor growth of GC. LLI internalizes into MFC cells to generate ROS causing immunogenic cell death (ICD). Simultaneously, liposome-deliver Liproxstatin-1 effectively inhibits the ferroptosis of tumor neutrophils. LLI-based immunogenic PDT and neutrophil-targeting immunotherapy synergistically boost the anti-PD-1 treatment to elicit potent TME and systemic antitumor immune response with abscopal effects. In conclusion, LLI holds great potential for GC immunotherapy.

[Genes for cellulose-degradation and their expression conditions in Chaetomium globosum NK102].

OBJECTIVE: Use of renewable plant biomass is an active area in current biotechnology research. This report explores the cellulose-degradation system and the factors affecting cellulase gene expression in Chaetomium globosum NK102. METHODS: In the sequenced genome, we identified 10 cellulase genes by sequence homology alignment. We used a high-throughput sequencing technology (RNA-Seq) to monitor the differential expression of the genes under different culture conditions. RESULTS: We observed that cellulase activity increased with time in the fungal cultures. Accordingly, transcription of the genes encoding cellobiohydrolase, cellobiose dehydrogenase and endoglucanase (cbh1, cdh and eglI) was higher than the others. Expression of the transcriptional repressors, ACE I and CreA, decreased with the time, whereas expression of Hap2/3/5 complex was upregulated. In different carbon sources, cellulase activity and their gene transcription were repressed by glucose and were activated by cellobiose. Sorbitol had no significant effect. Interestingly, light affected positively the expression of these cellulase genes. CONCLUSION: Differential RNA-seq analysis can make preliminary exploration on the expression regulation of cellulase genes. Expression of the genes in C. globosum was determined by different culture conditions. This study has shown a molecular system of cellulose-degradation in C. globosum and provides information for interpretation of carbohydrate metabolism.

Proteomics and metabolomics analysis of the lignin degradation mechanism of lignin-degrading fungus Aspergillus fumigatus G-13.

Aspergillus fumigatus has the potential to degrade lignocellulosic biomass, but the degradation mechanism is not clear. The purpose of this study is to analyze the differential proteins and metabolites produced by Aspergillus fumigatus G-13 in the degradation of different lignin model compounds. Ferulic acid, sinapic acid, and p-coumaric acid were used as carbon sources. By controlling the culture conditions, and adding a cellulose co-substrate and an auxiliary carbon source, the enzymatic production law of three lignin model compounds degraded by Aspergillus fumigatus G-13 was investigated. Proteomics and metabolomics analysis were conducted for the two groups with the largest difference in enzyme activity expression. The results showed that a total of 1447 peptides were identified by proteomics analysis. Among them, 134 proteins were significantly changed, 73 proteins were up-regulated, and 61 proteins were down-regulated. The key proteins that degrade lignin model compounds are catechol dioxygenase, glutathione reductase, dextranase, isoamyl alcohol oxidase, glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase. Enrichment analysis of differential metabolite functions revealed that Aspergillus fumigatus G-13 is associated with several pathways related to the degradation of lignin. Among them, starch and sucrose metabolism, pentose phosphate pathway, glutathione metabolism, and the ortho-cleavage pathway of dihydroxylated aromatic rings are closely related to lignin degradation. The information presented in this paper will be helpful for future research on the degradation or depolymerization of natural lignocellulosic substrates.

[Two optimized transformation protocols for a cellulose-utilizing fungus Chaetomium globosum NK-102].

OBJECTIVE: We developed the transformation methods of the strain Chaetomium globosum NK-102. METHODS: We constructed plasmid pUCATPH-Pgap and compared the transformation efficiency with pUCATPH and pCM768. We established the PEG mediated protoplast transformation and Agrobacterium tumefaciens EHA105 mediated transformation methods. RESULTS: In protoplast approach, approximately 3 -5 transformants/microg DNA could be obtained. The highest efficiency of transformation was obtained by employing pUCATPH-Pgap. A. tumefaciens EHA105 successfully mediated T-DNA insertion into the genome of C. globosum NK-102 and the transformation rate was 3.2 x 10(2) transformants/10(7) spores. The transformants retained stable after generations. Southern blot analyses confirmed that the DNA had integrated into the chromosomal DNA of C. globosum NK-102. CONCLUSION: The transformation systems were good basis for selection of C. globosum mutant strains that effectively utilizing cellulose.

A Smart Multifunctional Nanoparticle for Enhanced Near-Infrared Image-Guided Photothermal Therapy Against Gastric Cancer.

BACKGROUND: Surgery is considered to be a potentially curative approach for gastric cancer. However, most cases are diagnosed at a very advanced stage for the lack of typical symptoms in the initial stage, which makes it difficult to completely surgical resect of tumors. Early diagnosis and precise personalized intervention are urgent issues to be solved for improving the prognosis of gastric cancer. Herein, we developed an RGD-modified ROS-responsive multifunctional nanosystem for near-infrared (NIR) imaging and photothermal therapy (PTT) against gastric cancer. METHODS: Firstly, the amphiphilic polymer was synthesized by bromination reaction and nucleophilic substitution reaction of carboxymethyl chitosan (CMCh) and 4-hydroxymethyl-pinacol phenylborate (BAPE). Then, it was used to encapsulate indocyanine green (ICG) and modified with RGD to form a smart multifunctional nanoparticle targeted to gastric cancer (CMCh-BAPE-RGD@ICG). The characteristics were determined, and the targeting capacity and biosafety were evaluated both in vitro and in vivo. Furthermore, CMCh-BAPE-RGD@ICG mediated photothermal therapy (PTT) effect was studied using gastric cancer cells (SGC7901) and SGC7901 tumor model. RESULTS: The nanoparticle exhibited suitable size (≈ 120 nm), improved aqueous stability, ROS-responsive drug release, excellent photothermal conversion efficiency, enhanced cellular uptake, and targeting capacity to tumors. Remarkably, in vivo studies suggested that CMCh-BAPE-RGD@ICG could accurately illustrate the location and margin of the SGC7901 tumor through NIR imaging in comparison with non-targeted nanoparticles. Moreover, the antitumor activity of CMCh-BAPE-RGD@ICG-mediated PTT could effectively suppress tumor growth by inducing necrosis and apoptosis in cancer cells. Additionally, CMCh-BAPE-RGD@ICG demonstrated excellent biosafety both in vitro and in vivo. CONCLUSION: Overall, our study provides a biocompatible theranostic nanoparticle with enhanced tumor-targeting ability and accumulation to realize NIR image-guided PTT in gastric cancer.

A new composite of graphene and molecularly imprinted polymer based on ionic liquids as functional monomer and cross-linker for electrochemical sensing 6-benzylaminopurine.

Molecularly imprinted polymers prepared using traditional functional monomers and cross-linkers exhibit slow binding kinetics, low electrocatalytic activity and adsorption capacity. Herein, we report a new composite of ionic liquid-based graphene and molecularly imprinted polymer (IL-GR-MIP) with high electrocatalytic activity and adsorption capacity to construct an effective electrochemical sensor for 6-benzylaminopurine (6-BAP). Our objective was to enhance the efficiency of the sensor by incorporating more IL in the MIP framework. We synthesized IL-GR-MIP using ionic liquid 1-vinyl-3-butylimidazolium tetrafluoroborate (IL1) as functional monomer, ionic liquid 1,4-butanediyl-3,3'-bis-l-vinylimidazolium dibromide (IL2) as cross-linker, 6-BAP as template, and GR as supporter. IL-GR-MIP was characterized by Fourier transform infrared spectroscopy, thermal gravimetric analysis, Raman spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscope. Compared with GR-MIP composites based on methacrylic acid or IL1 as functional monomer, N, N'-methylenebisacrylamide and ethylene glycol dimethacrylate as cross-linker, the IL-GR-MIP (prepared with ionic liquids as functional monomer and cross-linker) sensor exhibited highest peak current for 6-BAP. The results indicate the ability of IL2 as cross-linker to enhance electrocatalytic activity and adsorption capacity for 6-BAP of IL-GR-MIP. Under the optimized conditions, the peak current of IL-GR-MIP sensor was linear to 6-BAP concentration in the range of 0.5-50 µM with a detection limit of 0.2 µM (S/N = 3). The IL-GR-MIP sensor exhibited good selectivity with the anti-interference ability of 1000-fold ascorbic acid in 6-BAP determination. Furthermore, we demonstrated practical applicability of IL-GR-MIP sensor in detecting 6-BAP in real samples with satisfactory results.

Determination of melamine and cyromazine in milk by high performance liquid chromatography coupled with online solid-phase extraction using a novel cation-exchange restricted access material synthesized by surface initiated atom transfer radical polymerization.

A novel strong-cation-exchange restricted access material has been synthesized by atom transfer radical polymerization (ATRP). In the synthesis, poly(3-sulfopropyl methacrylate-co-ethylene dimethacrylate), [p(SPM/EDMA)] was grafted on the silica by surface-initiated ATRP first. The poly(glycerol mono-methacrylate) [pGMMA] was then immobilized on the external surface, which created a chemical diffusion barrier for protein exclusion. The resulting Sil-g-p(SPM/EDMA)-g-pGMMA has both functions of protein exclusion and cation exchange, exhibiting the property of cation-exchange restricted access material. The application of Sil-g-p(SPM/EDMA)-g-pGMMA has been studied by the determination of melamine and cyromazine in bovine milk using the online solid-phase extraction/HPLC method. In the process, the Sil-g-p(SPM/EDMA)-g-pGMMA was used for the sample pre-treatment and a HILIC column was employed as the analytical column. The method has shown good accuracy, precision and low limits of detections. The result demonstrated that the Sil-g-p(SPM/EDMA)-g-pGMMA can be used for the cation extraction from biological samples by direct HPLC injection.

Cloning, expression, and characterization of miR058 and its target PPO during the development of grapevine berry stone.

Polyphenol oxidases catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms. They are key enzymes playing a significant role during the synthesis of lignin. The inhibition of the synthesis of lignin in grapevine can cause seedless grapevine berry development. In this study, grapevine PPO (Vv-PPO) was predicted as the target gene of Vv-miR058 by bioinformatics analysis, and it was further cloned and its homologous conservation in various plants was analyzed. The expression profiles of miR058 and its target Vv-PPO were detected by qRT-PCR in peel, pulp and seeds of three grapevine cultivars and Vv-PPO was expressed in an opposite variation way with Vv-miR058 where both of them could be detected, suggesting that Vv-miR058 can play an important role by regulating the expression of Vv-PPO. In addition, the potential target gene Vv-PPO for Vv-miR058 was verified by RLM-RACE. This result would be helpful in theoretical basis for further research and seedless grapevine berry production.

[Research on population structure and distribution characteristic of indigenous microorganism in post-polymer-flooding oil reservoir].

Denaturing gradient gel electrophoresis (DGGE) method and principal component analysis (PCA) method were used to analyze the structures of microorganism population in injection wells and production wells of a post-polymer-flooding oil reservoir in Daqing oil field. The results showed that the dominant species in injection wellhead were aerobic bacteria Pseudomonas and Acinenobacter. Facultative anaerobic bacteria Enterbacter was the dominant bacteria in near area of injection wells. Bacteria detected in production wells included Thauera, Clostridia, Pseudomonas, Petrobacter and some uncultured bacteria. Methanosaeta turned out to be the only archaea detected in injection wells, which was an aceticlastic methane-producing archaeon. Archaea detected in production wells consisted of Methanomicrobium, Methanospirillum and Methanobacterium. In general, aerobic bacteria, facultative anaerobe, and strictly anaerobic bacteria distributed successively from injection wells to production wells in this block. The dominant populations of archaea were different between injection wells and production wells, while were influenced by different environments and microbial metabolism products.