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.

Spirocyclohexadienone-Type Neolignans with Neuroprotective and Neurite Outgrowth Enhancing Activities from Magnolia liliiflora.

Three rare spirocyclohexadienone-type neolignans, magnoflorins A-C (1-3), and three known analogs (4-6), were isolated from the leaves of Magnolia liliiflora. Magnoflorin D (4) was obtained from natural resources for the first time. The chemical structures and absolute configurations of 1-4 were elucidated through detailed analysis of HR-ESI-MS, IR, 1 H, 13 C, and 2D NMR, and ECD experiments. The absolute configuration of 5 were characterized by X-ray crystallography in present study. Moreover, compounds 4 and 5 displayed moderate neuroprotective activity against corticosterone-induced PC12 cells injury at 20 µM with cell viability of 71.5±0.99 % and 73.0±1.42 %, respectively, compared to the model group with 60.83±0.93 %. Compound 6 could enhance neurite outgrowth of nerve growth factor (NGF)-induced PC12 cells at 10 µM with the differentiation rate of 11.98 %, compared with 20.49 % of 50 ng/ml NGF.

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri.

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

Preparation of Lignin Carbon/Zinc Oxide Electrode Material and Its Application in Supercapacitors.

In this paper, carbon/zinc oxide (LC/ZnO) composites were successfully synthesized and characterized by X-ray powder diffraction, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, Raman, thermogravimetry, and N2 adsorption-desorption, and tested by electrochemical performance. Studies have shown that the morphology of LC/ZnO composites is that lignin pellets are embedded in ZnO microplates. The lignin carbon in the composites mainly exists in an amorphous structure, and the specific surface area and pore channels of metal oxides are increased by the presence of lignin carbon. The electrochemical performance test shows that the carbonization temperature of LC/ZnO with the highest specific capacitance is 550 °C, and the capacitance retention rate reaches 96.74% after 1000 cycles of testing, indicating that the composite material has good cycle stability. Compared with the control group, it is found that the specific capacitance of LC/ZnO-550 °C is 2.3 times and 1.8 times that of ZnO-550 °C and LC-550 °C, respectively. This shows that during the electrochemical test, the lignin carbon and the metal oxide promote each other and act synergistically. In addition, the composite material exhibits the characteristics of a pseudo-capacitance capacitor, indicating that the redox reaction occurred in the electrochemical performance test.

Fabrication of vascularized and scaffold-free bone tissue using endothelial and osteogenic cells differentiated from bone marrow derived mesenchymal stem cells.

Over-dependence on existing synthetic scaffolds and insufficient vascularization limit the development of tissue engineered bone (TEB). The purpose of this study is to fabricate vascularized and scaffold-free bone tissue using cell sheet technology and to assess its feasibility to repair critical-sized calvarial defects in rats. Firstly, the pre-vascularized cell sheet was formed by seeding BMSC-derived endothelial cells (ECs) on an undifferentiated BMSCs cell sheet layer in vitro. After 3 days of co-culture, ECs migrated and rearranged to form lumens on the BMSC sheet. Secondly, osteogenic cell sheet was formed by inducing osteogenic differentiation of high density BMSCs. Then, the pre-vascularized cell sheet was stacked on BMSC-derived osteogenic cell sheet to fabricate a scaffold-free construct for bone regeneration. Finally, the scaffold-free construct with both angiogenic and osteogenic potential was implanted into critical-sized calvarial defects in adult Wistar rats. Results showed that more functional perfused blood vessels and new bone tissue formed in the pre-vascularized group than that in the controls (both empty and non-pre-vascularized cell sheet group). This study indicates a new promising strategy for bone tissue regeneration.

Engineering biomimetic periosteum with ß-TCP scaffolds to promote bone formation in calvarial defects of rats.

BACKGROUND: There is a critical need for the management of large bone defects. The purpose of this study was to engineer a biomimetic periosteum and to combine this with a macroporous ß-tricalcium phosphate (ß-TCP) scaffold for bone tissue regeneration. METHODS: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were harvested and cultured in different culture media to form undifferentiated rBMSC sheets (undifferentiated medium (UM)) and osteogenic cell sheets (osteogenic medium (OM)). Simultaneously, rBMSCs were differentiated to induced endothelial-like cells (iECs), and the iECs were further cultured on a UM to form a vascularized cell sheet. At the same time, flow cytometry was used to detect the conversion rates of rBMSCs to iECs. The pre-vascularized cell sheet (iECs/UM) and the osteogenic cell sheet (OM) were stacked together to form a biomimetic periosteum with two distinct layers, which mimicked the fibrous layer and cambium layer of native periosteum. The biomimetic periostea were wrapped onto porous ß-TCP scaffolds (BP/ß-TCP) and implanted in the calvarial bone defects of rats. As controls, autologous periostea with ß-TCP (AP/ß-TCP) and ß-TCP alone were implanted in the calvarial defects of rats, with a no implantation group as another control. At 2, 4, and 8 weeks post-surgery, implants were retrieved and X-ray, microcomputed tomography (micro-CT), histology, and immunohistochemistry staining analyses were performed. RESULTS: Flow cytometry results showed that rBMSCs were partially differentiated into iECs with a 35.1% conversion rate in terms of CD31. There were still 20.97% rBMSCs expressing CD90. Scanning electron microscopy (SEM) results indicated that cells from the wrapped cell sheet on the ß-TCP scaffold apparently migrated into the pores of the ß-TCP scaffold. The histology and immunohistochemistry staining results from in vivo implantation indicated that the BP/ß-TCP and AP/ß-TCP groups promoted the formation of blood vessels and new bone tissues in the bone defects more than the other two control groups. In addition, micro-CT showed that more new bone tissue formed in the BP/ß-TCP and AP/ß-TCP groups than the other groups. CONCLUSIONS: Inducing rBMSCs to iECs could be a good strategy to obtain an endothelial cell source for prevascularization. Our findings indicate that the biomimetic periosteum with porous ß-TCP scaffold has a similar ability to promote osteogenesis and angiogenesis in vivo compared to the autologous periosteum. This function could result from the double layers of biomimetic periosteum. The prevascularized cell sheet served a mimetic fibrous layer and the osteogenic cell sheet served a cambium layer of native periosteum. The biomimetic periosteum with a porous ceramic scaffold provides a new promising method for bone healing.

Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration.

Engineering three-dimensional (3D) vascularized constructs remains a challenge due to the inability to form rich microvessel networks. In this study we engineered a prevascularized 3D cell sheet construct for tissue regeneration using human bone marrow-derived mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells as cell sources. hMSCs were cultured to form a thick cell sheet, and human umbilical vein endothelial cells (HUVECs) were then seeded on the hMSCs sheet to form networks. The single prevascularized HUVEC/hMSC cell sheet was folded to form a 3D construct by a modified cell sheet engineering technique. In vitro results indicated that the hMSCs cell sheet promoted the HUVECs cell migration to form networks in horizontal and vertical directions. In vivo results showed that many blood vessels grew into the 3D HUVEC/hMSC cell sheet constructs after implanted in the subcutaneous pocket of immunodeficient mice. The density of blood vessels in the prevascularized constructs was higher than that in the nonprevascularized constructs. Immunohistochemistry staining further showed that in vitro preformed human capillaries in the prevascularized constructs anastomosed with the host vasculature to form functional blood vessels. These results suggest the promising potential of this 3D prevascularized construct using hMSCs cell sheet as a platform for wide applications in engineering vascularized tissues.