Ganoderma lucidum is a Chinese traditional medicine with various bioactivities. However, the impacts of a Ganoderma lucidum mycelium-fermented liquid (GLFL), as a promising alternative product, on the gut microbes and cardiovascular risk factors have not been explored in humans to date. In this study, the composition of polysaccharides in GLFL was analyzed, and eight volunteers were fed with GLFL to investigate its influence on gut microbes and cardiovascular risk factors. High-throughput 16S rRNA gene sequencing technique was utilized to investigate the effects of GLFL on the diversity of the bacterial communities and the composition of gut microbiota. The results demonstrated that GLFL significantly altered β diversity and several phyla of gut microbiota in volunteers. Moreover, GLFL provided protection to humans by promoting the growth of probiotics, incorporating genus Lactobacillus (p < 0.05), and reducing pathogens containing genus Campylobacter and Aggregatibacter (p < 0.05); however, GLFL also had detrimental effects as it increased the population of Firmicutes/Bacteroidetes and opportunistic pathogens including genus Acinetobacter, Pseudomonas, Serratia, Stenotrophomonas, Peptococcus and S24-7 (p < 0.05) and reduced that of probiotics genus Lactococcus. Finally, we found that GLFL reduced plasma low-density lipoprotein cholesterol (LDL-c) in volunteers. This is helpful for further understanding the effect of Ganoderma lucidum or its related products on human body.

Ganoderma lucidum is a Chinese traditional medicine with various bioactivities. However, the impacts of a Ganoderma lucidum mycelium-fermented liquid (GLFL), as a promising alternative product, on the gut microbes and cardiovascular risk factors have not been explored in humans to date. In this study, the composition of polysaccharides in GLFL was analyzed, and eight volunteers were fed with GLFL to investigate its influence on gut microbes and cardiovascular risk factors. High-throughput 16S rRNA gene sequencing technique was utilized to investigate the effects of GLFL on the diversity of the bacterial communities and the composition of gut microbiota. The results demonstrated that GLFL significantly altered β diversity and several phyla of gut microbiota in volunteers. Moreover, GLFL provided protection to humans by promoting the growth of probiotics, incorporating genus Lactobacillus (p < 0.05), and reducing pathogens containing genus Campylobacter and Aggregatibacter (p < 0.05); however, GLFL also had detrimental effects as it increased the population of Firmicutes/Bacteroidetes and opportunistic pathogens including genus Acinetobacter, Pseudomonas, Serratia, Stenotrophomonas, Peptococcus and S24-7 (p < 0.05) and reduced that of probiotics genus Lactococcus. Finally, we found that GLFL reduced plasma low-density lipoprotein cholesterol (LDL-c) in volunteers. This is helpful for further understanding the effect of Ganoderma lucidum or its related products on human body.

•Two COFs materials were fabricated to be photosensitizers.•The material functional property can be ameliorated by modifying the building block.•Two COFs exhibit good performance in photodynamic inactivation of bacteria.With the increase of antibiotic resistances in microorganisms, photodynamic inactivation (PDI) as a clinically proven antibacterial therapy is gaining increasing attention in recent years due to its high efficacy. Herein, we reported two covalent organic frameworks (COFs) materials, namely COFs-Trif-Benz and COF-SDU1, as effective type-II photosensitizers for photodynamic inactivation of bacteria. COFs-Trif-Benz and COF-SDU1 are synthesized through a facile solvothermal reaction between tri-(4-formacylphenoxy)-1,3,5-triazine (trif) and benzidine or p-phenylenediamine with high yield. Their highly ordered and porous structures were confirmed by Fourier transform infrared (FT-IR) spectra, solid state 13C CP/MAS NMR spectrum, powder X-ray diffraction (PXRD) and Brunauer–Emmett–Teller (BET) analyses. The electronic absorption spectra and electrochemical experiments revealed that the extensive π-conjugation over COFs-Trif-Benz and COF-SDU1 greatly enhance their absorbance capability for visible light and make them have a lower band gap. The photocatalytic antibacterial assay was studied against both Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli O86:B7 (E. coli O86) bacteria. Two materials can kill more than 90% bacteria at concentrations of 100 μg mL− 1 after 60–90 min of illumination. Thus, both COFs are effective photosensitizers. Mechanism investigation revealed the antibacterial characteristics of the COFs-Trif-Benz and COF-SDU1 can generate reactive oxygen species (ROS) by energy transfer to molecular oxygen (3O2) to produce a highly reactive singlet oxygen (1O2). Hence, the two materials during the photodynamic were mainly via mechanism type II.Download high-res image (211KB)Download full-size image

Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, albeit with weak immunogenicity. In this study, the xenoantigen l-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via the anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity toward A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancers of tumor associated antigens in the development of tumor immunotherapies.

•A UTP-glucose-1-phosphate uridylyltransferase (GalUSpe4) and a galactokinase (GalKSpe4) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system.•Six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion.•This is the first time it has been reported that UDP-Glc can be synthesized in a simple one-pot three-enzyme synthesis reaction system.A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.Graphical abstractEnzymatic synthesis of UDP-Glc/Gal and their derivatives by three-enzyme reaction system.

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Galactokinases (GalKs) have attracted significant research attention for their potential applications in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 presents a remarkably broad substrate range including 14 diverse natural and unnatural sugars. TLC and MS studies revealed that GalKSpe4 had relaxed activity towards galactose derivatives with modifications on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable. More interestingly, GalKSpe4 can phosphorylate l-mannose in moderate yield (43%), while other l-sugars such as l-Gal cannot be recognized by this enzyme. These results are very significant because there is rarely enzyme reported that can phosphorylate such uncommon substrates as l-mannose.

A β1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion, either Mn2+ or Mg2+, for its activity and is active between pH 6.0–8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase, benzyl β-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility of WbgO.The β1,3-galactosyltransferase (WbgO) involved in Escherichia coli O55:H7 O-antigen biosynthesis was recombinant expressed and characterized. Incorporated with LgtA catalyzed N-acetylglucosamine transferring reaction, lacto-N-tetraose was synthesized with recombinant WbgO.

Interferon-β has anti-viral, anti-proliferation and multifunctional immunomodulatory activities and shows promising clinical effects for treatment of inflammatory disorders. The recombinant human interferon-β (huIFN-β) 1b was expressed in the food-grade lactic acid bacterium, Lactococcus lactis, using a nisin-controlled gene expression system. huIFN-β production from recombinant strains (with and without LEISSTCDA propeptide) was approximately 21 and 7 μg l−1, respectively. Moreover, 95% (former strain) and 88% (latter strain) of total recombinant proteins were secreted into the culture medium. The biological activities of huIFN-β from recombinant strains revealed similar antiviral activities of 107 I.U. mg−1. These results demonstrate the potential application of recombinant strains as a food grade vehicle to deliver bioactive huIFN-β in vivo.

Using Agrobacterium mediated transient expression method, plant bivalent expression vector pBI121 containing GUS as a report gene was transformed into lettuce (Lactuca sativa). Through designed orthogonal analysis, intact lettuce leaves infiltrated with 200 μM acetosyringone and 0.8 OD600 bacterial suspensions under vacuum for 30 min, then co-cultured at 24 °C for 6 ds produced a maximum GUS protein of 2.5% TSP with 21.39 nmol mg−1 min−1 MU activity, which was 19 times of the control (1.31 nmol mg−1 min−1 MU). Employed these optimized conditions HuIFN-beta was expressed in lettuce leaves. Western blot and antivirus bioactivity analyses confirmed the HuIFN-beta achieved by agrobacterium infiltration had a high biological activity (3.1 × 104 IU/mL). To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.