The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

A Novel Mechanically Robust and Biodegradable Egg White Hydrogel Membrane by Combined Unidirectional Nanopore Dehydration and Annealing.

A homogeneous egg white obtained by high-speed shearing and centrifugation was dehydrated into a fragile and water-soluble egg white glass (EWG) by unidirectional nanopore dehydration (UND). After EWG annealing, it can become an egg white hydrogel membrane (EWHM) that is water-insoluble, flexible, biocompatible, and mechanically robust. Its tensile strength, elongation at break, and the swelling ratio are about 5.84 MPa, 50-110%, and 60-130%, respectively. Protein structure analysis showed that UND caused the rearrangement of the protein molecules to form EWG with random coil and α-helix structures. The thermal decomposition temperature of the EWG was 309.25 °C. After EWG annealing at over 100 or 110 °C for 1.0 h or 45 min, the porous network EWHM was mainly composed of ß-sheet structures, and the thermal decomposition temperature increased to 317.25-318.43 °C. Their 12-day residues in five proteases ranged from 1% to 99%, and the order was pepsin > neutral protease > papain > trypsin > alkaline protease. Mouse fibroblast L929 cells can adhere, grow, and proliferate well on these EWHMs. Therefore, the combined technology of UND and annealing for green and novel processing of EWHM has potential applications in the field of biomimetic and biomedical materials.

Identification of a novel laccase gene EuLAC1 and its potential resistance against Botrytis cinerea.

In this study, a novel laccase gene, EuLAC1, was cloned from Eucommia ulmoides Oliver (E. ulmoides). An overexpression vector harboring the EuLAC1 was constructed and introduced into the tobacco (Nicotiana tabacum cv. Xanthi). The laccase activity, resistance to Botrytis cinerea (B. cinerea) and lignin level in wild-type and transgenic plants were thereafter investigated. Interestingly, the transgenic tobacco displayed a significantly higher laccase activity and resistance to gray mold as compared to the wild-type tobacco. Additionally, the lignin contents in the leaves and stems of the transgenic tobacco were significantly higher in comparison to the wild-type tobacco. Scanning electron microscopy was used to observe the cross sections of wild-type and transgenic tobacco stems and it was noted that the cell wall near the xylem catheter of the transgenic tobacco was substantially thicker and the outline clearer than that of the wild-type. Thus, the EuLAC1 gene can significantly increase laccase activity and lignin content in tobacco, leading to an increase in the physical defenses, thereby increasing tobacco resistance to gray mold.

A neonate with hand, foot, and mouth disease complicated with brainstem encephalitis and pulmonary edema:A complete recovery.

Hand, foot, and mouth disease (HFMD) with serious complications and fatal cases have been reported over the last decade worldwide. The authors report a rare case of HFMD in a neonate complicated with brainstem encephalitis and pulmonary edema. She had fever, lethargy, dyspnea. Physical examination revealed shock signs, fine rales on both lungs, absent Moro reflex. The patient had a rapidly progressive course with seizures, coma, no spontaneous breathing, chemosis. There were some vesicles on left sole and red maculopapular rashes on perianal skin. She had a history of exposure to HFMD. Fecal sample was positive for EV71 RNA by real-time PCR. Chest X-rays showed bilateral pulmonary infiltrates. MRI of the brain showed significant hypointensity in the brainstem on T1WI and hyperintensity on T2WI. She recovered well. This case highlights severe HFMD in neonates is rare. Medical history and physical examination are important in making diagnosis.

Efficacy and safety of combinations of H1 antihistamines in the treatment of urticaria: A scoping review.

The efficacy and safety of combining H1 antihistamines (AHs) for treating urticaria are currently unclear. This scoping review aims to provide a comprehensive overview of the evidence regarding the efficacy and safety of H1 AH combinations in the management of urticaria up to May 2023. The search encompassed databases such as PubMed, Web of Science, the Cochrane Central Register of Controlled Trials, and the China Biological Medicine Database. The inclusion criteria comprised randomised controlled trials (RCTs), non-randomised trials (NRTs), case reports, and case series focusing on urticaria treatment. Initially screening 12,887 studies, this review ultimately selected 109 studies involving 11,435 patients. These studies documented 43 different combination treatments across 11 types of urticaria. In comparison to monotherapy, combination therapy exhibited superior efficacy in 94 studies that reported treatment efficacy. Regarding adverse drug reactions (ADRs), 67 studies disclosed ADR incidences, with combination therapy showing lower ADR rates in 32 studies. Additionally, 7 studies reported similar ADR rates between combination therapy and monotherapy with AHs. Common ADRs included symptoms such as drowsiness, nausea, fatigue, dry mouth, dizziness, and headache, while less frequent side effects encompassed hypotension, otitis media, polyuria, rhinorrhoea, abnormal liver function, and rash. ADR rates ranged from 0% to 21% in the treatment group, and from 0.5% to 75% in the control group. Importantly, patients generally tolerated these ADRs well, with symptoms resolving upon discontinuation of treatment. The study's findings suggest that combining AHs leads to enhanced efficacy and reduced safety risks compared to monotherapy in the context of urticaria treatment. These results advocate for considering combination therapy as a viable option in clinical practice, especially for chronic urticaria cases. Nonetheless, caution is advised, and close monitoring for potential ADRs is crucial during treatment.

An insight on egg white: From most common functional food to biomaterial application.

Natural egg white tis widely used as an ingredient in nutritional foods and for food processing. Due to its characteristic foaming, emulsification, adhesion, and gelation, and its heat setting, biocompatibility, and low cost, research into the application and development of egg white in biomaterials, especially medical biomaterials, have been receiving attention. The composition and characteristics of egg white protein, and the physical mixing and chemically cross-linking of egg white with other materials used to make degradable packaging films, bioceramics, bioplastics, biomimetic films, hydrogels, 3D scaffolds, bone regeneration, biopatterning, biosensors, and so forth, are reviewed in detail in this report. The novel egg white-based biomaterials in various forms and applications could be constructed mostly through physical treatments such as ultrasonic wave, ultraviolet, laser and other radiation or high-temperature calcination. Furthermore, the application and prospects for the use of egg white in biomaterials is also discussed.

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue.

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.

Charge-conversional zwitterionic copolymer as pH-sensitive shielding system for effective tumor treatment.

A novel pH-responsive gene delivery system for tumor acidity-targeted pDNA delivery is prepared by introducing a rapid charge-conversional zwitterionic copolymer to the positive surface of PEI/pDNA complexes through electrostatic interaction. The shielding system (OEAL) consists of oligoethylenimine (OEI), poly(l-aspartate) (PBLA), and poly(l-lysine) (PLL). The charge-conversional behavior of the OEAL/PEI/DNA ternary complex is evaluated by zeta potential assay. The surface charges of the complexes can change from negative to positive in the pH range of 7.4-6.8. Under a simulative in vivo environment, OEAL/PEI/DNA exhibits promotion of cellular uptake by tumor cells and enhanced gene transfection efficiency because of its good charge-conversional properties. Antitumor experiments further show that the pH-responsive charge-conversional system can mediate a therapeutic gene that can induce tumor apoptosis (pKH3-rev-casp-3) to achieve effective tumor inhibition. Accordingly, OEAL can be regarded as a promising tumor microenvironment-sensitive gene delivery shielding system for antitumor therapy. STATEMENT OF SIGNIFICANCE: This manuscript focused on the novel pH-responsive gene delivery system for tumor acidity-targeted pDNA delivery. The novel system is prepared by introducing a rapid charge-conversional zwitterionic copolymer, consisting of oligoethylenimine, poly(l-aspartate) and poly(l-lysine), to the positive surface of PEI/pDNA complexes. The surface charges of the complexes can change from negative to positive from pH 7.4 to 6.8. OEAL/PEI/DNA shows promoting cellular uptake by tumor cells and enhanced gene transfection efficiency. The antitumor experiments further show that the pH responsive charge conversional system can mediate pKH3-rev-casp-3 to achieve effective tumor inhibition. Accordingly, OEAL can be regarded as a promising tumor microenvironment sensitive gene delivery shielding system for antitumor therapy.

Effective tumor treatment by VEGF siRNA complexed with hydrophobic poly(amino acid)-modified polyethylenimine.

Two copolymers are designed and synthesised for siRNA delivery based on polyethylenimine by grafting hydrophilic acrylamide segments and hydrophobic poly(γ-benzyl L-glutamate). The amphiphilic PEI-PBLG/siRNA complex demonstrates high gene silencing efficiency in the absence or presence of 10 vol% and 50 vol% sera in vitro. The anti-tumor effects in vivo are evaluated in luciferase-bearing mice expressing CT26 tumors. PEI-PBLG/siVEGF complex provides a higher and more sustained suppressive effect by reducing VEGF mRNA expression in the tumors, leading to higher tumor growth inhibition efficacy. Further studies on the potential use of the PEI-PBLG carrier system in mediating the silencing of genes other than VEGF or in other tumor models are recommended.

A serum-tolerant hydroxyl-modified polyethylenimine as versatile carriers of pDNA/siRNA.

The polyethylenimine (PEI) derivatives (PTn) are prepared by treating PEI25k with Tris(hydroxymethyl) acrylamidomethane via the Michael addition. These PTns can effectively condense nucleic acids into nanosized particles with positive surface charges. The PTns show lower cytotoxicity and better serum-resistant capacity than PEI25k. Specially, the transfection efficiency of PT26/DNA is 29-fold higher than that of PEI25k in HeLa cells in serum-containing medium. The PTn/siRNA complexes show superior knockdown effect in CT26 cells in serum-containing medium. In addition, flow cytometry analysis shows that the PTns can efficiently mediate the entry of nucleic acids into the cell. Thus, PTns are potentially applicable as non-viral carriers of nucleic acids and warrant further development for use in gene therapy.

PEI conjugated gold nanoparticles: efficient gene carriers with visible fluorescence.

Low molecular weight polyethyleneimines were conjugated onto gold nanoparticles to form Au-PEI conjugates. Au-PEI is the first reported gene carrier which has both high transfection efficiency and strong fluorescence. Au-PEI showed higher transfection efficiency and it can be used for bioimaging because it can be detected by confocal laser scanning microscopy and in vivo bioimaging system.

Biodegradable mPEG-b-P(MCC-g-OEI) copolymers for efficient gene delivery.

Cationic polymers play an important role in gene delivery. To search for potential non-viral gene carriers, a series of biodegradable cationic polymers, poly(ethylene glycol)-block-poly(carbonates-graft-oligoethylenimine) [mPEG-b-P(MCC-g-OEI), PPO] copolymers, were synthesized by grafting different kinds of oligoethylenimine (OEI) to the same biodegradable backbone, a derivative of poly(ethylene glycol)-block-polycarbonates. The as-synthesized PPO copolymers were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gel permeation chromatography. Two of the as-synthesized PPO copolymers (PPO600 and PPO1800) could efficiently condense DNA into nanosized particles (100-140 nm) with positive surface charges when the PPO/DNA mass ratio is above 10:1. The cell toxicity and gene transfection evaluations show that PPO copolymers, especially PPO1800, which exhibits lower cytotoxicity and higher gene transfection efficiency than PEI25K in the absence and the presence of serum in the CHO and COS-7 cell lines, have great potential as non-viral gene carriers. The PPO1800 copolymer images obtained by confocal laser scanning microscopy prove that PPO copolymers could efficiently mediate the entry of plasmid DNA into cells. These results show that PPO copolymers may be potential non-viral gene carriers in future applications of gene therapy.

[Chondrogenic differentiation of adult human periodontal ligament stem cells in vitro].

OBJECTIVE: To isolate and cultivate human periodontal ligament stem cells (PDLSC) and to investigate the feasibility of PDLSC in vitro differentiation into chondrogenic phenotype. METHODS: Periodontal tissue was obtained from healthy young human teeth extracted for orthodontic purposes. PDLSCs were isolated by single-colony selection and cultivated. PDLSC of passage 3 was plated at density of 1 x 10(7) cells/cm3 and induced with chondrogenic induction medium of DMEM containing TGF-beta1 (10 microg/L), IGF-1 (50 microg/L), dexamethasone (40 microg/L) and 10% FBS. In control group, the constructs were maintained in DMEM medium + 10% FBS. After 21 days induction, the results were evaluated by histology, histochemistry, immunohistochemistry and RT-PCR. RESULTS: The constructs in experimental group were smooth and relatively firm in texture after 3 weeks of culture. Toluidine blue staining showed the formation of distinct lacuna structure. Positive staining of type II collagen was also detected by immunohistochemistry and it was confirmed by RT-PCR. In contrast, in the control group, the constructs collapsed gradually, lacuna was barely detected in histology and type II collagen expression negative. CONCLUSIONS: Periodontal ligament contain stem cells can be isolated and cultivated. PDLSC have the potential of chondrogenic differentiation.