Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Structure-based antigenic epitope and PEGylation improve the efficacy of staphylokinase.

Staphylokinase (Sak) holds promise for use in thrombolytic therapy for acute myocardial infarction. However, its immunogenicity is a major disadvantage under clinical conditions. PEGylation has become a sophisticated method to decrease that immunogenicity. In this report, according predicted epitope from the active center, five residues, including Gly79, Leu82, Lys84, Ala97, and Arg104 have been mutant as cysteine for mono PEGylation, respectively. According to the relative immunogenicity of Sak or its PEGylation derivatives, the amount of specific anti-Sak IgG antibodies elicited by PEGylation proteins, including C79G, C82L, C84K, C97A, and C104R in BALB/c mice decreased by approximately 15-75% each. PEGylated Sak derivatives showed a decrease of up to 75% in the immune reactivity in PEG-Sak-C104R. Thrombelastography experiments showed that two PEG-conjugated derivatives, PEG-Sak-C97A (Ly30, 68.14 ± 2.51%) and PEG-Sak-C104R (Ly30, 66.49 ± 5.97%), the LY30 of PEG-Sak-C97A, and PEG-Sak-C104R produced values very similar to those of wild-type Sak. The fibrin plate assays showed the bioactivity of PEG-Sak-C104R to exhibit the most activity approximately as much as urokinase (diameter of halo pattern, 18.6 ± 1.06 mm) and tPA (diameter of halo pattern, 17.2 ± 0.49 mm). The Sak PEGylation derivative PEG-Sak-C104R was also selected for further in vivo activity experimentation. The thrombolytic ability of PEG-Sak-C104R is a little lower than wild-type Sak, whereas, this PEGylated protein retained high activity suitable for thrombolytic therapy. Collectively, with the in vivo and in vitro experiments, the present study suggests that site mutant PEGylation, PEG-Sak-C104R, is a suitable type of PEGylation for clinical applications. Further optimization would help maintain the bioactivity and decrease the immunogenicity of staphylokinase.

Antibacterial activity and mechanism of Ag/ZnO nanocomposite against anaerobic oral pathogen Streptococcus mutans.

Dental caries is a widespread disease mainly caused by the anaerobic oral pathogen Streptococcus mutans (S. mutans). Ag/ZnO nanocomposite is an efficient antibacterial agent because of its high antibacterial activity and low cytotoxicity. In this study, rod-like Ag/ZnO nanocomposite was synthesized through a deposition-precipitation method and characterized by X-ray diffraction, scanning electron microscopy and transmission electron microscopy. The activity of Ag/ZnO nanocomposite against S. mutans was evaluated by determining the minimal inhibitory concentration, minimum bactericidal concentration and growth inhibition curve. The results showed that Ag/ZnO nanocomposite displayed higher activity against S. mutans compared with pure ZnO nanorods. Moreover, the antibacterial mechanism was investigated by determining the bacterial membrane potential, release of K+, intracellular reactive oxygen generation and lipid peroxidation. Disruption of membrane function and oxidation of biomacromolecules played important role in the antibacterial action of Ag/ZnO nanocomposite. This work proposes a potentially effective dental antibacterial agent against the dental caries-causing S. mutans.

Fabrication of an exosome-loaded thermosensitive chitin-based hydrogel for dental pulp regeneration.

Injective thermosensitive hydrogels are considered promising scaffolds to trigger dental pulp regeneration in devitalized human teeth. In this study, we developed a hydroxypropyl chitin (HPCH)/chitin whisker (CW) thermosensitive hydrogel with enhanced mechanical properties and biological activities. Exosomes can serve as biomimetic tools for tissue engineering, but the rapid clearance of unconjugated exosomes in vivo limits their therapeutic effects. To address this challenge, exosomes were isolated from human pulp stem cells (hDPSCs) and directly embedded into the HPCH/CW pre-gel to form an exosome-loaded hydrogel (HPCH/CW/Exo). The exosome-loaded thermosensitive hydrogel can be easily injected into an irregular endodontic space and gelated in situ. In vitro cell experiments proved that the delivery of exosomes significantly improved the ability of hydrogels to promote odontogenesis and angiogenesis. Meanwhile, in vivo animal experiments revealed the formation of new dental pulp-like tissues in an implanted tooth root model. Therefore, the proposed hydrogel provides a great potential alternative to traditional root canal therapy in dental clinics.

Extrafibrillarly Demineralized Dentin Matrix for Bone Regeneration.

Dentin is a natural extracellular matrix, but its availability in bone grafting and tissue engineering applications is underestimated due to a lack of proper treatment. In this study, the concept of extrafibrillar demineralization is introduced into the construction of dentin-derived biomaterials for bone regeneration for the first time. Calcium chelating agents with large molecular weights are used to selectively remove the extrafibrillar apatite minerals without disturbing the intrafibrillar minerals within dentin collagen, resulting in the formation of an extrafibrillarly demineralized dentin matrix (EDM). EDM with distinctive nanotopography and bone-like mechanical properties is found to significantly promote cell adhesion, migration, and osteogenic differentiation in vitro while enhancing in vivo bone healing of rat calvarial defects. The outstanding osteogenic performance of EDM is further confirmed to be related to the activation of the focal adhesion-cytoskeleton-nucleus mechanotransduction axis. Overall, this study shows that extrafibrillar demineralization of dentin has great potential to produce hierarchical collagen-based scaffolds for bone regeneration, and this facile top-down fabrication method brings about new ideas for the biomedical application of naturally derived bioactive materials.

Pretreatment of Luzhou distiller's grains for feed protein production using crude enzymes produced by a synthetic microbial consortium.

Chinese distillers' grains (CDGs) have low fermentation efficiency due to the presence of lignocellulosic components, such as rice husk. In this study, a microbial consortium synthesized was used based on the "functional complementarity" principle to produce lignocellulolytic crude enzyme. The crude enzyme was used to hydrolyze CDGs. After enzymatic hydrolysis, lignocellulose was damaged to varying degrees and the crystallinity decreased. Subsequently, the feed protein was produced using yeast through two pathways. The results showed that the crude enzyme produced by the microbial consortium (comprising Trichoderma reesei, Aspergillus niger, and Penicillium) exhibited excellent enzymatic efficiency, yielding 27.88%, 19.64%, and 10.88% of reducing sugar, cellulose, and hemicellulose. The true protein content of CDGs increased by 53.49% and 48.35% through the first and second pathways, respectively. Notably, the second pathway demonstrated higher economic benefits to produce feed protein. This study provides a pathway for high-quality utilization of CDGs.

Proteomics of Streptococcus mutans to Reveal the Antibiofilm Formation Mechanism of Ag/ZnO Nanocomposites with Light-Emitting Diode Radiation.

INTRODUCTION: As a biofilm-associated disease, dental caries benefits from nanoparticle (NP)-based therapies. Streptococcus mutans (S. mutans) is a primary aetiologic agent for dental caries development. We successfully applied a synergistic therapy of Ag/ZnO nanocomposites combined with light-emitting diode (LED) radiation to inhibit S. mutans biofilms. However, the antibiofilm mechanism has not been fully elucidated, and little is known about the biofilm formation ability of bacteria that survive NP-based therapies. METHODS: This study explored the antibiofilm formation mechanism of this synergistic therapy by an integrated approach based upon proteomics. RESULTS: Synergistic therapy killed 99.8% of bacteria, while the biofilm formation ability of 0.2% surviving bacteria was inhibited. The proteomic responses of S. mutans to synergistic therapy were comprehensively characterized to unveil the mechanism of bacterial death and biofilm formation inhibition of the surviving bacteria. In total, 55 differentially expressed proteins (12 upregulated and 43 downregulated) were recorded. The bioinformatic analysis demonstrated that cellular integrity damage and regulated expression of structure-associated proteins were the main reasons for bacterial death. In addition, the proteomic study indicated the potential inhibition of metabolism in surviving bacteria and provided a biofilm-related network consisting of 17 differentially expressed proteins, explaining the multiantibiofilm formation actions. Finally, we reported and verified the inhibitory effects of synergistic therapy on sucrose metabolism and D-alanine metabolism, which disturbed the biofilm formation of surviving bacteria. CONCLUSION: Our findings demonstrated that synergistic therapy killed most bacteria and inhibited the surviving bacteria from forming biofilms. Furthermore, the antibiofilm formation mechanism was revealed by proteomics analysis of S. mutans after synergistic therapy and subsequent metabolic studies. Our success may provide a showcase to explore the antibiofilm formation mechanism of NP-based therapies using proteomic studies.

Effects of Ag/ZnO nanocomposite at sub-minimum inhibitory concentrations on virulence factors of Streptococcus mutans.

OBJECTIVE: Streptococcus mutans (S. mutans), the main pathogen of dental caries, could be well killed by Ag/ZnO nanocomposite. However, effects on virulence factors remain to be elucidated. This study investigated effects of Ag/ZnO at sub-minimum inhibitory concentrations (sub-MICs) on virulence factors of S. mutans and related genes expressions. DESIGN: Effects of Ag/ZnO on the growth of S. mutans was investigated by growth curves and MTT staining method. The influence of Ag/ZnO at sub-MICs on biofilm formation was measured by the crystal violet staining method and observed by a scanning electron microscopy. Adherence, cell-surface hydrophobicity, acidogenicity and extracellular polysaccharides (EPS) of S. mutans after treatment by Ag/ZnO at sub-MICs were also investigated. Virulence factors related genes expressions after treated by Ag/ZnO at 1/2 MIC was conducted by the quantitative real-time PCR (qRT-PCR) method. RESULTS: Sub-MICs of Ag/ZnO exhibited a dose-dependent inhibition on the virulence factors of S. mutans. Specially, Ag/ZnO at 1/2 MIC decreased 69.00 % biofilm formation, 31.78 % sucrose-independent and 48.08 % sucrose-dependent adherence, 69.44 % cell-surface hydrophobicity and 72.45 % water-soluble and 90.60 % water-insoluble EPS. Furthermore, the expression of virulence factors related genes was significantly suppressed by Ag/ZnO at 1/2 MIC. CONCLUSIONS: Ag/ZnO at sub-MICs inhibited multiple virulence factors of S. mutans through downregulating the related genes. Ag/ZnO nanocomposite could be used for prevention of dental caries at low dosage.

Rapid Antibiofilm Effect of Ag/ZnO Nanocomposites Assisted by Dental LED Curing Light against Facultative Anaerobic Oral Pathogen Streptococcus mutans.

The integration of nanomaterials with clinical therapeutic instruments is a promising approach to improve the effects of nanomaterials. We reported an efficient synergistic antibacterial strategy formed through the combination of Ag/ZnO nanocomposites with a light-emitting diode (LED) curing light, which is a commonly used small instrument in dental clinics. The as-designed integration depicted a significantly enhanced bactericidal effect on facultative anaerobic oral pathogen Streptococcus mutans (S. mutans) both in planktonic and biofilm phases over a very short irradiation time (≤5 min). Further study showed that the combination of LED and Ag/ZnO nanocomposites induced more ·OH and ·O2- generation, which is responsible for the enhanced antibacterial activity. Moreover, this combination could destroy S. mutans biofilm by killing the bacteria embedded within biofilm, inhibiting exopolysaccharide production and down-regulating the biofilm-related gene expression. Therefore, it is proposed that this combination could be applied in dental clinics to realize dental caries prevention and dental restoration simultaneously.

Strain distribution of repaired articular cartilage defects by tissue engineering under compression loading.

BACKGROUND: It is difficult to repair cartilage damage when cartilage undergoes trauma or degeneration. Cartilage tissue engineering is an ideal treatment method to repair cartilage defects, but at present, there are still some uncertainties to be researched in cartilage tissue engineering including the mechanical properties of the repaired region. METHODS: In this study, using an agarose gel as artificial cartilage implanted into the cartilage defect and gluing the agarose gel to cartilage by using the medical bio-adhesive, the full-thickness and half-thickness defects models of articular cartilage in vitro repaired by tissue engineering were constructed. Strain behaviors of the repaired region were analyzed by the digital correlation technology under 5, 10, 15, and 20% compressive load. RESULTS: The axial normal strain (Ex) perpendicular to the surface of the cartilage and lateral normal strain (Ey) as well as shear strain (Exy) appeared obviously heterogeneous in the repaired region. In the full-defect model, Ex showed depth-dependent strain profiles where maximum Ex occurs at the low middle zone while in the half-defect mode, Ex showed heterogeneous strain profiles where maximum Ex occurs at the near deep zone. Ey and Exy at the interface site of both models present significantly differed from the host cartilage site. Ey and Exy exhibited region-specific change at the host, interface, and artificial cartilage sites in the superficial, middle, and deep zones due to the artificial cartilage implantation. CONCLUSION: Both defect models of cartilage exhibited a heterogeneous strain field due to the engineered cartilage tissue implant. The abnormal strain field can cause the cells within the repaired area to enter complex mechanical states which will affect the restoration of cartilage defects.

Chlorhexidine-encapsulated mesoporous silica-modified dentin adhesive.

OBJECTIVES: This work aims to explore the feasibility of chlorhexidine-encapsulated mesoporous silica (CHX@pMSN) as a modifier of a commercial dental adhesive via the evaluation of physicochemical properties and antibacterial capabilities of adhesive-dentin interface. METHODS: Therapeutic adhesives were developed in the present study by incorporating CHX@pMSN into a commercial adhesive at four mass fractions (0, 1, 5 and 10 wt.%). The antibacterial capability on Streptococcus mutans (S. mutans) biofilm, conversion degree, adhesive morphology, microtensile bond strength (MTBS) and nanoleakage expression were evaluated comprehensively. RESULTS: MTT and CLSM evaluation showed that CHX@pMSN-doped adhesive inhibits S. mutans biofilm growth, while CHX is released from the modified adhesive continuously. The incorporation of CHX@pMSN did not affect immediate bond strength at the concentration of 1% and 5% (P > 0.05). Moreover, these bonds were mainly preserved in 5% CHX@pMSN group after one month of collagenase ageing. Meanwhile, CHX@pMSN-doped adhesive groups exhibited similar nanoleakage distribution compared with the control. CONCLUSION: This study showed that the 5% CHX@pMSN-modified adhesive achieved balance amongst unaffected immediate bonding strength, well-preserved bonds against collagenase ageing and effective inhibition of S. mutans biofilm growth. CLINICAL SIGNIFICANCE: CHX@pMSN-modified dentin adhesive can potentially extend the service life of adhesive restoration in clinic.

An alternative choice of lidocaine-loaded liposomes: lidocaine-loaded lipid-polymer hybrid nanoparticles for local anesthetic therapy.

PURPOSE: The skin permeation enhancement of local anesthetics by newer innovative nanotechnologies has been an appealing field recently. However, which nanocarrier is better for drug loading and has better stability? Therefore, the aim of our study was to compare two kinds of nanocarriers: liposomes and lipid-polymer hybrid nanoparticles (LPNs) for lidocaine (LA) delivery. METHODS: LA-loaded liposomes (LA-LPs) and LPNs (LA-LPNs) were prepared. Two kinds of nanocarriers were characterized in terms of particle size, zeta potential, drug encapsulation efficiency (EE), drug release, and stability. Their in vitro skin permeation was studied using a Franz diffusion cell mounted with depilated mouse skin in vitro. In vivo local anesthetic effects of LA containing formulations were evaluated by tail flick latency (TFL) test using a tail-flick measuring device. RESULTS: Compared with LA-LPs, LA-LPNs showed significantly better in vitro skin permeation ability and in vivo local anesthetic effects. CONCLUSION: The results demonstrated that LPNs could improve the efficacy of drugs to higher levels than LPs and free drugs, thus could serve as an effective drug system for LA loading for local anesthetic therapy.