Molybdenum disulfide nanosheets based non-oxygen-dependent and heat-initiated free radical nanogenerator with antimicrobial peptides for antimicrobial, biofilm ablation and wound healing.

Chronic refractory wounds caused by multidrug-resistant (MDR) bacterial and biofilm infections are a substantial threat to human health, which presents a persistent challenge in managing clinical wound care. We here synthesized a composite nanosheet AIPH/AMP/MoS2, which can potentially be used for combined therapy because of the photothermal effect induced by MoS2, its ability to deliver antimicrobial peptides, and its ability to generate alkyl free radicals independent of oxygen. The synthesized nanosheets exhibited 61 % near-infrared (NIR) photothermal conversion efficiency, marked photothermal stability and free radical generating ability. The minimal inhibitory concentrations (MICs) of the composite nanosheets against MDR Escherichia coli (MDR E. coli) and MDR Staphylococcus aureus (MDR S. aureus) were approximately 38 µg/mL and 30 µg/mL, respectively. The composite nanosheets (150 µg/mL) effectively ablated >85 % of the bacterial biofilm under 808-nm NIR irradiation for 6 min. In the wound model experiment, approximately 90 % of the wound healed after the 4-day treatment with the composite nanosheets. The hemolysis experiment, mouse embryonic fibroblast (MEFs) cytotoxicity experiment, and mouse wound healing experiment all unveiled the excellent biocompatibility of the composite nanosheets. According to the transcriptome analysis, the composite nanosheets primarily exerted a synergistic therapeutic effect by disrupting the cellular membrane function of S. aureus and inhibiting quorum sensing mediated by the two-component system. Thus, the synthesized composite nanosheets exhibit remarkable antibacterial and biofilm ablation properties and therefore can be used to improve wound healing in chronic biofilm infections.

Pluronic-Based Nanoparticles for Delivery of Doxorubicin to the Tumor Microenvironment by Binding to Macrophages.

The active targeting drug delivery system based on special types of endogenous cells such as macrophages has emerged as a promising strategy for tumor therapy, owing to its tumor homing property and biocompatibility. In this work, the active tumor-targeting drug delivery system carrying doxorubicin-loaded nanoparticles (DOX@MPF127-MCP-1, DMPM) on macrophage (RAW264.7) surfaces via the mediation of interaction with the CCR2/MCP-1 axis was exploited. Initially, the amphiphilic block copolymer Pluronic F127 (PF127) was carboxylated to MPF127 at the hydroxyl terminus. Subsequently, MPF127 was modified with MCP-1 peptide to prepare MPF127-MCP-1 (MPM). The DOX was wrapped in MPM to form DMPM nanomicelles (approximately 100 nm) during the self-assembly process of MPM. The DMPM spontaneously bound to macrophages (RAW264.7), which resulted in the construction of an actively targeting delivery system (macrophage-DMPM, MA-DMPM) in vitro and in vivo. The DOX in MA-DMPM was released in the acidic tumor microenvironment (TME) in a pH-responsive manner to increase DOX accumulation and enhance the tumor treatment effect. The ratio of MA-DMPM homing reached 220% in vitro compared with the control group, indicating that the MA-DMPM was excellently capable of tumor-targeting delivery. In in vivo experiments, nonsmall cell lung cancer cell (NCI-H1299) tumor models were established. The results of the fluorescence imaging system (IVIS) showed that MA-DMPM demonstrated tremendous tumor-targeting ability in vivo. The antitumor effects of MA-DMPM in vivo indicated that the proportion of tumor cell apoptosis in the DMPM-treated group was 63.33%. The findings of the tumor-bearing mouse experiment proved that MA-DMPM significantly suppressed tumor cell growth, which confirmed its immense potential and promising applications in tumor therapy.

A pH-responsive polymer-coated CaO2as oxygen-generating nanoparticlein situfor enhanced chemo-photodynamic synergistic therapy against tumors.

Photodynamic therapy (PDT) has emerged as an efficient strategy for tumor treatment. However, Insufficient amounts of inherent hypoxia and intrinsic hydrogen peroxide (H2O2) in the tumor microenvironment severely constrained PDT, as oxygen is the critical substrate for photosensitivity reaction. Here, a pH-responsive H2O2and O2self-supplying hybrid nanoparticle was designed. Through, the calcium peroxide (CaO2) as carriers loading a chemotherapeutic drug a photosensitizer 5,10,15,20-tetrakis(4-aminophenyl) porphyrin (TAPP) and doxorubicin (DOX), was covered with polyacrylic acid (PAA) to build up a feature material DOX-TAPP-CaO2@OA@PAA (denoted as DTCOP) through the reverse microemulsion method. In the acidic tumor microenvironment conditions exposing the water-sensitive CaO2nanocore to generate hydrogen peroxide (H2O2) and O2, the self-supplied O2alleviates hypoxia to enhance the PDT, and releasing DOX and TAPP. Synthetic characterization shows that the succeeded synthesized Nanocarriers could effectively carry DOX and TAPP to the tumor site and release O2at the low pH of TME. And the experimental results demonstrated that this interpose exogenous oxygen strategy is efficient at inhibition of tumor growth bothin vitroandin vivo. The nanocomposite exhibits excellent biocompatibility and the ability to inhibit tumor growth and has significant potential for the treatment of hypoxic tumors.

Synthesis of a Two-Dimensional Molybdenum Disulfide Nanosheet and Ultrasensitive Trapping of Staphylococcus Aureus for Enhanced Photothermal and Antibacterial Wound-Healing Therapy.

Photothermal therapy has been widely used in the treatment of bacterial infections. However, the short photothermal effective radius of conventional nano-photothermal agents makes it difficult to achieve effective photothermal antibacterial activity. Therefore, improving composite targeting can significantly inhibit bacterial growth. We inhibited the growth of Staphylococcus aureus (S. aureus) by using an extremely low concentration of vancomycin (Van) and applied photothermal therapy with molybdenum disulfide (MoS2). This simple method used chitosan (CS) to synthesize fluorescein 5(6)-isothiocyanate (FITC)-labeled and Van-loaded MoS2-nanosheet hydrogels (MoS2-Van-FITC@CS). After modifying the surface, an extremely low concentration of Van could inhibit bacterial growth by trapping bacteria synergistically with the photothermal effects of MoS2, while FITC labeled bacteria and chitosan hydrogels promoted wound healing. The results showed that MoS2-Van-FITC@CS nanosheets had a thickness of approximately 30 nm, indicating the successful synthesis of the nanosheets. The vitro antibacterial results showed that MoS2-Van-FITC with near-infrared irradiation significantly inhibited S. aureus growth, reaching an inhibition rate of 94.5% at nanoparticle concentrations of up to 100 µg/mL. Furthermore, MoS2-Van-FITC@CS could exert a healing effect on wounds in mice. Our results demonstrate that MoS2-Van-FITC@CS is biocompatible and can be used as a wound-healing agent.

Acid-Sensitive Nanoparticles Based on Molybdenum Disulfide for Photothermal-Chemo Therapy.

The combination of multiple treatments has recently been investigated for tumor treatment. In this study, molybdenum disulfide (MoS2) with excellent photothermal conversion performance was used as the core, and manganese dioxide (MnO2), which responds to the tumor microenvironment, was loaded on its surface by liquid deposition to form a mesoporous core-shell structure. Then, the chemotherapeutic drug Adriamycin (DOX) was loaded into the hole. To further enhance its water solubility and stability, the surface of MnO2 was modified with mPEG-NH2 to prepare the combined antitumor nanocomposite MoS2@DOX/MnO2-PEG (MDMP). The results showed that MDMP had a diameter of about 236 nm, its photothermal conversion efficiency was 33.7%, and the loading and release rates of DOX were 13 and 65%, respectively. During in vivo and in vitro studies, MDMP showed excellent antitumor activity. Under the combined treatment, the tumor cell viability rate was only 11.8%. This nanocomposite exhibits considerable potential for chemo-photothermal combined antitumor therapy.

A bone-targeting drug delivery vehicle of a metal-organic framework conjugate with zoledronate combined with photothermal therapy for tumor inhibition in cancer bone metastasis.

Chemotherapy is a conventional treatment method for metastatic bone cancer, but it has limitations, such as lower drug-targeting of bone tissues and serious side effects. Bone metastasis almost always occurs in advanced cancer, and most patients in this period have strong drug resistance, which further worsens the curative effect. To address the above-mentioned difficulties, a drug delivery platform is proposed in this paper that accomplishes the bone-targeting of drugs to efficiently inhibit tumors. First, the anti-cancer drugs 5-fluorouracil (5-Fu) and indocyanine green (ICG) were loaded into a zeolitic imidazolate framework (ZIF-90) to form 5-Fu/ICG@ZIF-90. Polyethylene glycol with zoledronic acid (ZOL) was encapsulated using 5-Fu/ICG@ZIF-90 to synthesize 5-Fu/ICG@ZIF-90-PEG-ZOL nanoparticles, which showed dimensional stability, good thermal stability, and bone-targeting ability. Second, the in vitro anti-cancer activity of the designed platform was investigated using cytotoxicity, apoptosis, live-dead staining, cell cycle, and cell ultrathin section analysis. The results indicated that the nanoparticles inhibited MCF-7 cell activity when chemotherapy was combined with PTT. Finally, H&E staining and TUNEL detection were performed in mouse organs and tumors. The nanoparticles combined with photothermal therapy (PTT) and triggered by near-infrared irradiation induce apoptosis of tumor cells in vivo, displaying a better efficacy of combined chemotherapy and photothermal therapy. Experiments conducted on the 5-Fu/ICG@ZIF-90-PEG-ZOL nanoparticles demonstrated their promising performance for cancer bone metastasis inhibition.

Gold nanorod-loaded thermosensitive liposomes facilitate the targeted release of ruthenium(II) polypyridyl complexes with anti-tumor activity.

Ruthenium(II) polypyridyl complexes (Ru) show high anti-tumor activity, but their poor solubility and low biocompatibility impede their use in anti-tumor therapy. Here,we circumvented the problem of low solubility by encapsulating the Ru in thermosensitive liposomes (LTSLs) and used gold nanorods (Au NRs) modified on the surface of the liposomes to permit the precise release of Ru at the tumor site. A facile and simple method was developed to synthesize Ru-loaded Au NR-decorated LTSL (Au@LTSL-Ru NPs). The loaded Au NRs improved the anti-tumor effect of Ru and enhanced the photothermal therapeutic properties of the nanosystem. A characterization experiment indicated that the average particle size of Au@LTSL-Ru was approximately 300 nm and that the Au NRs were successfully modified on the surface of LTSL. In thein vitroanti-tumor test, Au@LTSL-Ru and NIR significantly inhibited the proliferation of SGC-7901 cells. The IC50value of Au@LTSL-Ru + NIR was 7.1 ± 1.2µM (13µg ml-1), and the inhibition rate was greater than 90% when the concentration reached 30µg ml-1.In vivostudies revealed that Au@LTSL-Ru and NIR had a significant inhibitory effect on subcutaneous tumor tissues derived from SGC-7901 cells. Analysis of histopathology and immunocytotoxicity indicated that Au@LTSL-Ru has fewer side effects and high biocompatibility. Our results confirm that Au@LTSL-Ru can effectively inhibit tumor growth and aid the development of Ru for use in the thermal response in anti-tumor activity research.

The Effects of Luminescent CdSe Quantum Dot-Functionalized Antimicrobial Peptides Nanoparticles on Antibacterial Activity and Molecular Mechanism.

BACKGROUND: With the development of bacterial resistance, the range of effective antibiotics is increasingly becoming more limited. The effective use of nanoscale antimicrobial peptides (AP) in therapeutic and diagnostic methods is a strategy for new antibiotics. METHODS: Combining both AP and cadmium selenide (CdSe) into a composite material may result in a reagent with novel properties, such as enhanced antibacterial activity, fluorescence and favorable stability in aqueous solution. RESULTS: AP-loaded CdSe NPs (AP-CdSe NPs) showed strong antibacterial activity against multidrug-resistant (MDR) Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro and in vivo. Colony-forming unit (CFU) and minimum inhibitory concentration (MIC) assays showed that AP-CdSe NPs have highly effective antibacterial activity. The quantitative analysis of apoptosis by flow cytometry analysis further confirmed that MDR E. coli and S. aureus treated with AP-CdSe NPs had death rates of 98.76% and 99.13%, respectively. Also, AP-CdSe NPs was found to inhibit bacterial activity in an in vivo bacteremia model in mice infected with S. aureus. In addition, the antibacterial mechanism of AP-CdSe NPs was determined by RNA sequencing analysis. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed the molecular mechanism of the antibacterial effect of AP-CdSe NPs. Importantly, histopathology analysis, and hematological toxicity analysis indicated that AP-CdSe NPs had few side effects. CONCLUSION: These results demonstrate that AP loaded on CdSe NPs had a higher water solubility, bioavailability and antibacterial effect compared with raw AP. This study reports findings that are helpful for the design and development of antibacterial treatment strategies based on AP.

De novosynthesis of pH-responsive, self-assembled, and targeted polypeptide nano-micelles for enhanced delivery of doxorubicin.

Doxorubicin (DOX) is a commonly used anticancer drug, but it is inefficient as a therapeutic due to a lack of targeting. Peptide-tuned self-assembly of DOX offers a strategy to improve targeting for greater efficacy. In this work, we designed and prepared an amphiphilic tumor cell-targeting peptide, P14 (AAAAFFFHHHGRGD), able to encapsulate DOX by self-assembly to form tumor cell-targeting and pH-sensitive nano-micelles. The results showed a critical P14-micelle concentration of 1.758 mg l-1and an average particle size of micelles of 121.64 nm, with entrapment and drug-loading efficiencies of 28.02% ± 1.35% and 12.06% ± 0.59%, respectively. The prepared micelles can release 73.52 ± 1.27% DOX within 24 h in pH 4.5 medium, and the drug cumulative release profile of micelles can be described by the first-order model. Compared with free DOX, the micelles exhibited an increased ability to inhibit tumor cell growth and cause tumor apoptosisin vitro, with IC50values of DOX and P14-DOX micelles against human breast cancer cells (MCF-7) of 0.91 ± 0.07 and 0.75 ± 0.06µg ml-1, respectively, and cellular apoptotic rates of DOX and P14-DOX micelles of 70.3% and 42.4%, respectively. Cellular uptake experiments revealed high concentrations of micelles around and inside MCF-7 cells, demonstrating that micelles can target tumor cells. These results indicate the excellent potential for the application of this amphiphilic peptide as a carrier for small-molecule drugs and suggest a strategy for the design of effective anti-tumor drugs.

An auto-inducible expression and high cell density fermentation of Beefy Meaty Peptide with Bacillus subtilis.

Currently, some cases about the expression of flavor peptides with microorganisms were reported owing to the obvious advantages of biological expression over traditional methods. However, beefy meaty peptide (BMP), the focus of umami peptides, has neither been concerned in its safe expression nor its overproduction in fermenter. In this study, multi-copy BMP (8BMP) was successfully auto-inducibly expressed and efficiently produced in Bacillus subtilis 168. First, 8BMP was successfully auto-inducibly expressed with srfA promoter in B. subtilis 168. Further, the efficient production of 8BMP was researched in a 5-L fermenter: the fermentation optimized by Pontryagin's maximum principle obtained the highest 8BMP yield (3.16 g/L), which was 1.2 times and 1.8 times than that of two-stage feeding cultivation (2.67 g/L) and constant-rate feeding cultivation (1.75 g/L), respectively. Overall, the auto-inducible expression of 8BMP in B. subtilis and fermentation with Pontryagin's maximum principle are conductive for overproduction of BMP and other peptides.

Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans.

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes α-phenylalanine to ß-phenylalanine, and could be used to synthesize unnatural ß-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pam), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, 15N-(2S)-α-phenylalanine, (2S)-(3-2H2)-α-phenylalanine and (2S,3S)-[2,3-2H2]-α-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of α-phenylalanine. The PaPAM shuttles the α-NH2 of α-phenylalanine to ß site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to α site to produce ß-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.

One-Pot Enzymatic Synthesis of D-Arylalanines Using Phenylalanine Ammonia Lyase and L-Amino Acid Deaminase.

The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction solution could be converted into the D-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH3BH3. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (eeD)) of D-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize D-arylalanine with different groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

Preparation of a tumor-targeted drug-loading material, amphiphilic peptide P10, and analysis of its anti-tumor activity.

A new tumor-targeted drug-loading material, the amphiphilic peptide DGRGGGAAAA (P10) was designed and synthesized, and its self-assembly behavior, drug-loading effects and in vitro characteristics were studied. P10 was synthesized by solid-state synthesis and doxorubicin (DOX) was loaded via dialysis. P10 and DOX were mixed with a mass ratio of 6:1 to form regular round spheres. The interconnection between groups was analyzed spectroscopically and the sphere morphology was studied with SEM and a zeta particle size analyzer. Fluorescence spectroscopy was used to analyze the ability of P10 to form micelles and the efficiency of micelle entrapment, and the drug-loading ratio and drug release characteristics were detected. Finally, the in vitro antitumor activity of P10 was studied with HeLa cells as a model. The results showed that P10's critical micelle concentration (CMC) value and its average grain diameter were approximately 0.045 mg/L and 500 nm. The micelle entrapment ratio and drug-loading ratio were 23.011 ± 2.88 and 10.125 ± 2.62%, respectively, and the in vitro drug-releasing properties of P10 were described by the Zero-order model and the Ritger-Peppas model. Compared with DOX, P10-DOX had a higher tumor cell inhibition ratio and a dose-effect relationship with concentration. When P10-DOX's concentration was 20 µg/mL, the inhibition ratio was 44.17%. The new amphiphilic peptide designed and prepared in this study could be a tumor-targeted drug-loading material with better prospects for application. In this paper, a new tumor-targeted drug-loading material, the amphiphilic peptide DGRGGGAAAA (P10) is designed and synthesized, and its self-assembly behavior, drug-loading effects and in vitro characteristics are studied, providing a theoretical basis and design ideas for further studies and the development of targeted drug-loading materials on tumor cells.

The Soluble and Particulate Form of Alginates Positively Regulate Immune Response.

BACKGROUND: Alginate materials have been widely employed for biomedical applications ranging from wound healing to cancer treatment. However, how alginate materials affect the immune system is largely unknown. OBJECTIVE: To explore the impact of alginate materials on immune system. METHODS: The effect of three types of alginate materials, low viscosity, high viscosity and particulate alginate, were examined by both in vivo and in vitro analyses. C57BL/6J (B6) mice were treated with alginate and peripheral blood was tested by ELISA for cytokine production. Dendritic cells, macrophages and splenocytes isolated from mice were analyzed for the response to alginate treatment. Administration of alginates by intra lymph node injection (I.L.N.) yielded more potent cytokines productions than other injection routes. RESULTS: Alginate materials did not affect the viability of lymphocytes. Particulate alginate induced the most potent inflammatory reaction as determined by the production of cytokines, such as, IL-1ß, IL-8, TNF-α and IFN-γ. Low viscosity and particulate alginates are more effective than high viscosity alginates in activating dendritic cells as indicated by the expression of dendritic cells surface markers (CD80, CD86 and CD40). Similarly, the level of G-CSF was slightly higher in particulate alginate treated macrophages. CONCLUSION: Alginate materials could affect immune response through different ways, including promoting inflammatory cytokine production, and activating dendritic cells. Therefore, alginate materials, especially in particulate form, have the potential to be applied in inflammation related diseases.

One step synthesis of unnatural ß-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high ß-regioselectivity.

Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield ß-phe. However, the final product mixture consists of both α- and ß-phe owing to low regioselectivity, which is still a challenge to synthesize highly pure ß-phe. Therefore, a modified TcPAM with high ß-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn458 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroamination of t-CA and as promising candidates for mutagenesis to enhance ß-selectivity and decrease α-selectivity. The Asn458 and Leu108 residues were mutated to yield variant TcPAM-Asn458Phe/Leu108Glu, and the ß-selectivity was approximately 5.2-fold higher than that of wild-type TcPAM, while α-selectivity decreased to 68%, and the percentage of ß-phe in the product mixture increased from 42% to 83%. In addition, the mutant was applied to synthesize ß-arylalanines using substituent t-CA as a substrate. The regioselectivity was also affected by the substituent groups at the phenyl ring of t-CA with respect to their electronic properties and position, and the 4-methoxy and methyl substituent t-CA were transferred into ß-arylalanines. The conversion rate also exceeded 90%. In summary, the engineered TcPAM proved to be useful for one-step asymmetric amination of t-CA and its derivatives to synthesize highly pure ß-arylalanines.

Nano-graphene oxide-manganese dioxide nanocomposites for overcoming tumor hypoxia and enhancing cancer radioisotope therapy.

While radiotherapy (RT) is commonly used in clinics for cancer treatment, the therapeutic efficiency is not satisfactory owing to the existence of the hypoxic tumor microenvironment which seriously affects the efficiency of RT. Herein, we design polyethylene glycol (PEG)-modified reduced nano-graphene oxide-manganese dioxide (rGO-MnO2-PEG) nanocomposites to trigger oxygen generation from H2O2 to reduce the tumor hypoxic microenvironments. We use the radioisotope, 131I labeled rGO-MnO2-PEG nanocomposites as therapeutic agents for in vivo tumor radioisotope therapy (RIT), achieving excellent tumor killing and further enhancing the therapeutic efficiency of RIT. More importantly, the dissolution of MnO2 under acidic conditions and the redox process during the catalytic pathway of H2O2 decomposition in the cellular microenvironment direct to the production of an enormous amount of Mn2+ which has been used as a contrast agent for magnetic resonance imaging (MRI). Our proposed work provides a strategy to trigger oxygen formation via an internal stimulus to enhance imaging-guided RIT efficiency.

Recent advances in enhanced enzyme activity, thermostability and secretion by N-glycosylation regulation in yeast.

Yeast has been increasingly used as a host for the expression of enzymes. Compared to other expression systems, the yeast expression system has many advantages including its suitability for large-scale fermentation and its ability to modify enzymes. When expressed in yeast, many recombinant enzymes are N-glycosylated, and this may play an important role in their activity, thermostability and secretion. Although the mechanism underlying this process is not clear, the regulation of N-glycosylation by introducing or eliminating N-glycosylation at specific sites has developed into an important strategy for improving the production or catalytic properties of recombinant enzymes. In this review, we summarize the recent advances in understanding the effects of N-glycosylation on the expression and characteristics of recombinant enzymes, and discuss novel strategies for regulating N-glycosylation in yeast. We hope that this review will help improve the understanding of the expression and the catalytic properties of N-glycosylated proteins.

Bioconversion of farnesol and 1,4-dihydroxy-2-naphthoate to menaquinone by an immobilized whole-cell biocatalyst using engineered Elizabethkingia meningoseptica.

Menaquinone (MK) has important applications in the pharmaceutical and food industries. To increase the production rate (QP) of MK-4, we developed a straightforward biotransformation method for MK-4 synthesis directly from its precursors 1,4-dihydroxy-2-naphthoate (DHNA) and farnesol using whole cells of genetically engineered Elizabethkingia meningoseptica. Results showed that MK-4 can be produced directly from farnesol and DHNA using both free and immobilized FM-D198 cells. MK-4 yield peaked at 29.85 ± 0.36 mg/L in the organic phase and 24.08 ± 0.33 mg/g DCW after 12 h of bioconversion using free cells in a two-phase conversion system. MK-4 yield reached 26.34 ± 1.35 mg/L and 17.44 ± 1.05 mg/g DCW after 8 h using immobilized cells. Although this yield was lower than that using free cells, immobilized cells can be re-used for MK-4 production via repeated-batch culture. After ten batch cultures, efficient MK-4 production was maintained at a yield of more than 20 mg/L. After optimizing the catalysis system, the MK-4 yield reached 26.91 ± 1.27 mg/L using the immobilized cells and had molar conversion rates of 58.56 and 76.90% for DHNA and farnesol, respectively.

Alginate Particles with Ovalbumin (OVA) Peptide Can Serve as a Carrier and Adjuvant for Immune Therapy in B16-OVA Cancer Model.

BACKGROUND Alginate is a natural polysaccharide obtained from brown algae and has been shown to have numerous applications in biomedical science, such as wound healing, delivery of bioactive agents, and cell transplantation. Ovalbumin (OVA) peptide 323-339 has been reported to be involved in immune response.  MATERIAL AND METHODS This work investigated the use of alginate particles as a carrier and adjuvant for the immune therapy of cancer. Alginate particles loaded with OVA peptide were produced via emulsion. A tumor model was established in C57BL/6J mice via subcutaneous injection of 3×105 B16-OVA tumor cells. The effect of alginate/OVA peptide on cell viability was analyzed by use of the CCK-8 assay kit. Activation of macrophages was examined by checking cell surface makers CD40 and CD86 by FACs. RESULTS Alginate/OVA peptide inhibited tumor progression more effectively than using the peptide alone. The viability and uptake study illustrated that this particle is safe and non-toxic. The activation study demonstrated that alginate particles can promote the activation of surface markers on macrophages. ELISA assay showed that the particles with peptide can promote the secretion of inflammatory and effector cytokines from macrophages.  CONCLUSIONS This study demonstrated that alginate has dual functions in immune therapy of cancer, serving both as a carrier and an adjuvant.

Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from Anabaena variabilis by Modification of Center-Near Surface Residues.

Phenylalanine ammonia lyase from Anabaena variabilis (Av-PAL) is a candidate for the treatment of phenylketonuria (PKU). However, Av-PAL shows its optimal pH at 8.5 and maintains only 70% of its highest activity when pH decreases to 7.3-7.4 (the condition of human plasma). The objective of the study was to shift its optimal pH by mutating surface amino acid residues which interact with the general base Tyr78. Based on the crystal structure and the online program GETAREA, we selected five sites: Asn69, Glu72, Glu75, Asn89, and Val90. Removing negative charges or introducing positive charges near the general base Tyr78 by mutation, the pH optima were successfully shifted to more acidic range. Especially, the pH optima of E75A, E75L, and E75Q were shifted to 7.5 with 35, 30, and 24% higher specific activities than that of the wild, respectively. Half-lives of E75L and E75Q at 70 °C prolonged to 190 and 180 min from 130 min of the wild, respectively. In addition, the higher resistance to a low pH of 3.5 and protease made E75L a candidate for oral medicine of PKU. This work would improve the therapeutic prospect of Av-PAL and provide guidance in modulating optimal pH of enzymes.