Compatible and Incompatible Mycorrhizal Fungi With Seeds of Dendrobium Species: The Colonization Process and Effects of Coculture on Germination and Seedling Development.

Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

Prophylactic vaccine delivery systems against epidemic infectious diseases.

Prophylactic vaccines have evolved from traditional whole-cell vaccines to safer subunit vaccines. However, subunit vaccines still face problems, such as poor immunogenicity and low efficiency, while traditional adjuvants are usually unable to meet specific response needs. Advanced delivery vectors are important to overcome these barriers; they have favorable safety and effectiveness, tunable properties, precise location, and immunomodulatory capabilities. Nevertheless, there has been no systematic summary of the delivery systems to cover a wide range of infectious pathogens. We herein summarized and compared the delivery systems for major or epidemic infectious diseases caused by bacteria, viruses, fungi, and parasites. We also included the newly licensed vaccines (e.g., COVID-19 vaccines) and those close to licensure. Furthermore, we highlighted advanced delivery systems with high efficiency, cross-protection, or long-term protection against epidemic pathogens, and we put forward prospects and thoughts on the development of future prophylactic vaccines.

Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method.

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

Transport of a graphene nanosheet sandwiched inside cell membranes.

The transport of nanoparticles at bio-nano interfaces is essential for many cellular responses and biomedical applications. How two-dimensional nanomaterials, such as graphene and transition-metal dichalcogenides, diffuse along the cell membrane is, however, unknown, posing an urgent and important issue to promote their applications in the biomedical area. Here, we show that the transport of graphene oxides (GOs) sandwiched inside cell membranes varies from Brownian to Lévy and even directional dynamics. Specifically, experiments evidence sandwiched graphene-cell membrane superstructures in different cells. Combined simulations and analysis identify a sandwiched GO-induced pore in cell membrane leaflets, spanning unstable, metastable, and stable states. An analytical model that rationalizes the regimes of these membrane-pore states fits simulations quantitatively, resulting in a mechanistic interpretation of the emergence of Lévy and directional dynamics. We finally demonstrate the applicability of sandwiched GOs in enhanced efficiency of membrane-specific drug delivery. Our findings inform approaches to programming intramembrane transport of two-dimensional nanomaterials toward advantageous biomedical applications.

Improving adjuvanticity of quaternized chitosan-based microgels for H5N1 split vaccine by tailoring the particle properties to achieve antigen dose sparing effect.

In this study, we developed the quaternized chitosan microgels without chemical crosslinking as an adjuvant of H5N1 split vaccine. The microgels with pH-sensitivity, positive surface charge and good biocompatibility, have been demonstrated in favor of enhancing both humoral and cellular immune response. However, the detailed mechanism of the chitosan-based microgels to enhance antigen specific immune responses remains unclear. Therefore, we prepared the quaternized chitosan microgels with well defined quaternization degrees (QDs, 20-80%) and particle sizes (800nm-5µm) by the premix membrane emulsification technique, and investigated the effect of quaternization degree (QD) and size on the adjuvanticity of microgels. Results suggested that microgels with relatively smaller size (807nm) and moderate quaternization degree (QD 41% and 60%) were favorable for a maximum immune response. The mechanism was studied and explained by examining the characteristics of microgels and investigating the stimulation of bone-marrow derived dendritic cells (BMDCs). Moreover, they induced significantly stronger immune responses at lower antigen doses (known as antigen sparing effect) compared to aluminum adjuvant. These data indicated that a maximum immune response can be achieved by controlling properties of chitosan microgels, which also could serve as a significant guidance for rational design of chitosan-based particle adjuvant.

Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening.

Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 µM without apparent toxicity (CC50 > 200 µM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 µM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 µM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 µM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.

The potential adjuvanticity of quaternized chitosan hydrogel based microparticles for porcine reproductive and respiratory syndrome virus inactivated vaccine.

Infectious diseases possess a big threat to the livestock industry worldwide. Currently, inactivated veterinary vaccines have attracted much attention to prevent infection due to their safer profile compared to live attenuated vaccine. However, its intrinsic poor immunogenicity demands the incorporation of an adjuvant. Mineral oil based adjuvant (Montanide™ ISA206) was usually used to potentiate the efficacy of veterinary vaccines. However, ISA206 could not induce robust cellular immune responses, which was very important in controlling virus replication and clearing the infected cells. Moreover, mineral oil would result in severe side effects. To improve both the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (PRRSV) inactivated vaccine, we developed pH-sensitive and size-controllable quaternized chitosan hydrogel microparticles (Gel MPs) without using chemical cross linking agent. Gel MPs, ionic cross-linked with glycerophosphate (GP), were biocompatible and could efficiently adsorb the inactivated PRRSV vaccine with a loading capacity of 579.05µg/mg. After intramuscular immunization in mice, results suggested that Gel MPs elicited significantly higher cell-mediated immune responses and comparable humoral immune responses compared to ISA 206. Regarding the biocompatibility, safety and effectiveness, Gel MPs would be a promising candidate to enhance the efficacy of veterinary vaccine.

Recent Studies of Pickering Emulsions: Particles Make the Difference.

In recent years, emulsions stabilized by micro- or nanoparticles (known as Pickering emulsions) have attracted much attention. Micro- or nanoparticles, as the main components of the emulsion, play a key role in the preparation and application of Pickering emulsions. The existence of particles at the interface between the oil and aqueous phases affects not only the preparation, but also the properties of Pickering emulsions, affording superior stability, low toxicity, and stimuli-responsiveness compared to classical emulsions stabilized by surfactants. These advantages of Pickering emulsions make them attractive, especially in biomedicine. In this review, the effects of the characteristics of micro- and nanoparticles on the preparation and properties of Pickering emulsions are introduced. In particular, the preparation methods of Pickering emulsions, especially uniform-sized emulsions, are listed. Uniform Pickering emulsions are convenient for both mechanistic research and applications. Furthermore, some biomedical applications of Pickering emulsions are discussed and the problems hindering their clinical application are identified.

Chitosan-mediated formation of biomimetic silica nanoparticles: an effective method for manganese peroxidase immobilization and stabilization.

Our work here, for the first time, reported the use of chitosan-mediated biomimetic silica nanoparticles in enzyme immobilization. In order to make clear the relationship among silicification process, silica nanoparticle structure and immobilized enzyme activity, a mechanism of chitosan-mediated silicification using sodium silicate as the silica source was primarily evaluated. Chitosan was demonstrated effectively to promote the silicification not only in accelerating the aggregation rate of sodium silicate, but also in templating the formation of silica nanoparticles. Although the whole biomimetic silicification process contained polycondensation-aggregation-precipitation three stages, the elemental unit in precipitated silica was confirmed to be nanoparticles with 100 nm diameter regardless of the chitosan and silicate concentration used. Furthermore, the effect of enzyme on silicification process was also investigated. The introducing of manganese peroxidase (MnP) to silica precursor solution had no obvious effect on the silicification rate and nanoparticle morphology. The residual activity and embedding rate of immobilized MnP were 64.2% and 36.4% respectively under the optimum conditions. In addition, compared to native MnP, the MnP embedded in chitosan/silica nanoparticles exhibited improved stability against organic solvent and ultrasonic wave. After ultrasonic treatment for 20 min, 77% of the initial activity was remained due to the protective effect of chitosan/silica nanoparticles, while native MnP lost almost all of its original activity.

Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis.

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

Porous TiO2 materials through Pickering high-internal phase emulsion templating.

We report a facile method for preparing porous structured TiO2 materials by templating from Pickering high-internal phase emulsions (HIPEs). A Pickering HIPE with an internal phase of up to 80 vol %, stabilized by poly(N-isopropylacrylamide)-based microgels and TiO2 solid nanoparticles, was first formulated and employed as a template to prepare the porous TiO2 materials with an interconnected structure. The resultant materials were characterized by scanning electron microscopy, X-ray diffraction, and mercury intrusion. Our results showed that the parent emulsion droplets promoted the formation of macropores and interconnecting throats with sizes of ~50 and ~10 µm, respectively, while the interfacially adsorbed microgel stabilizers drove the formation of smaller pores (~100 nm) throughout the macroporous walls after drying and sintering. The interconnected structured network with the bimodal pores could be well preserved after calcinations at 800 °C. In addition, the photocatalytic activity of the fabricated TiO2 was evaluated by measuring the photodegradation of Rhodamine B in water. Our results revealed that the fabricated TiO2 materials are good photocatalysts, showing enhanced activity and stability in photodegrading organic molecules.

Novel vaccine delivery system induces robust humoral and cellular immune responses based on multiple mechanisms.

Aiming to enhance the immunogenicity of H5N1 split vaccine, the development of a novel antigen delivery system based on quaternized chitosan hydrogel microparticles (Gel MPs) with multiple mechanisms of immunity enhancement is attempted. Gel MPs based on ionic cross-linking are prepared in a simple and mild way. Gel MPs are superior as a vaccine delivery system due to their ability to: 1) enhance cellular uptake and endosomal escape of antigens in dendritic cells (DCs); 2) significantly activate DCs; 3) form an antigen depot and recruit immunity cells to improve antigen capture. Further in vivo investigation shows that Gel MPs, in comparison to aluminum salts (Alum), LPS, and covalent cross-linking quaternized chitosan MPs (GC MPs), induce higher humoral and cellular immune responses with a mixed Th1/Th2 immunity. In conclusion, these results demonstrate that Gel MPs are efficient antigen delivery vehicles based on multiple mechanisms to enhance both humoral and cellular immune responses against H5N1 split antigen.

Multiscale evaluation of pore curvature effects on protein structure in nanopores.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14-120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

Harnessing the potential of halogenated natural product biosynthesis by mangrove-derived actinomycetes.

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH2-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.

Nanoparticles-based multi-adjuvant whole cell tumor vaccine for cancer immunotherapy.

Whole cell tumor vaccine (WCTV), as a potential treatment modality, elicits limited immune responses because of the poor immunogenicity. To address this issue, researchers have attempted to transduce a cytokine adjuvant into tumor cells, but these single-adjuvant WCTVs curtail the high expectations. In present study, we constructed a multi-adjuvant WCTV based on the nanoparticles modified with cell penetrating peptide, which could facilitate the transportation of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2) into tumor cells. After inactivation, as-designed multi-adjuvant WCTV exhibited programmed promotions on DC recruitment, antigen presentation, and T-cell activation. In vivo evaluations demonstrated the satisfactory effects on tumor growth suppression, metastasis inhibition, and recurrence prevention. Therefore, the nanoparticles-based multi-adjuvant WCTV may serve as a high-performance treatment for anti-tumor immunotherapy.

Codelivery of mTERT siRNA and paclitaxel by chitosan-based nanoparticles promoted synergistic tumor suppression.

Clinical applications of siRNA are being hindered by poor intracellular uptake and enzymatic degradation. To address these problems, we devised an oral delivery system for telomerase reverse transcriptase siRNA using N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles (HNP). Both the porous structure and the positive charge of HNP facilitated siRNA encapsulation. The outer coating of HTCC not only protected siRNA from enzymatic degradation, but also improved siRNA permeability in intestine tract. In vivo and in vitro experiments proved that HNP could effectively deliver siRNA to lesion site and further into tumor cells. On the basis of confirming the antitumor activity of HNP:siRNA, we continued to encapsulate a hydrophobic chemotherapeutic drug-paclitaxel (PTX) into HNP to form a "two-in-one" nano-complex (HNP:siRNA/PTX). We demonstrated that HNP:siRNA/PTX could simultaneously ferry siRNA and PTX into tumor cells and increase drug concentration, which, in particular, was much more effective in tumor suppression than that of traditional cocktail therapy. These results suggested that the HNP, as a powerful delivery system for both siRNA and chemotherapeutic drug, would have a far-reaching application in human cancer therapy.

Thermal-sensitive hydrogels as nasal vaccine delivery systems.

The National Key Laboratory of Biochemical Engineering established in 1995 is affiliated with the Institute of Process Engineering, Chinese Academy of Sciences (CAS), and located in Zhong-guan-cun (Beijing, China). The National Key Laboratory of Biochemical Engineering is working towards developments in the fields of bio-reaction, bioseparation and bio-formulation, by chemical and material methods. Over the last 5 years, approximately 200 scientists and students have worked at the laboratory, and published over 400 articles. Numerous universities, companies and institutes have established cooperative relationships with this laboratory, and over 70 cooperative research programs with other researchers have been conducted in the past few years.

Microcosmic mechanisms for protein incomplete release and stability of various amphiphilic mPEG-PLA microspheres.

The microcosmic mechanisms of protein (recombinant human growth hormone, rhGH) incomplete release and stability from amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres were investigated. PELA with different hydrophilicities (PELA-1, PELA-2, and PELA-3) based on various ratios of mPEG to PLA were employed to prepare microspheres exhibiting a narrow size distribution using a combined double emulsion and premix membrane emulsification method. The morphology, rhGH encapsulation efficiency, in vitro release profile, and rhGH stability of PELA microspheres during the release were characterized and compared in detail. It was found that increasing amounts of PLA enhanced the encapsulation efficiency of PELA microspheres but reduced both the release rate of rhGH and its stability. Contact angle, atomic force microscope (AFM), and quartz crystal microbalance with dissipation (QCM-D) techniques were first combined to elucidate the microcosmic mechanism of incomplete release by measuring the hydrophilicity of the PELA film and its interaction with rhGH. In addition, the pH change within the microsphere microenvironment was monitored by confocal laser scanning microscopy (CLSM) employing a pH-sensitive dye, which clarified the stability of rhGH during the release. These results suggested that PELA hydrophilicity played an important role in rhGH incomplete release and stability. Thus, the selection of suitable hydrophilic polymers with adequate PEG lengths is critical in the preparation of optimum protein drug sustained release systems. This present work is a first report elucidating the microcosmic mechanisms responsible for rhGH stability and its interaction with the microspheres. Importantly, this research demonstrated the application of promising new experimental methods in investigating the interaction between biomaterials and biomacromolecules, thus opening up a range of exciting potential applications in the biomedical field including drug delivery and tissue regeneration.