Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-class Ⅰ complex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-class Ⅰ and Ⅱ molecules and for prediction of the binding ability of the peptide HLA-class Ⅰ complexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-class Ⅰ and Ⅱ molecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC50<500 nmol/L to HLA-class Ⅰ and Ⅱ molecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding geno-types.Peptides with affinity IC50<500 nmol/L had an affinity core sequence overlap of no less than 8 amino acids,and can be priori-tized for candidate T cell epitopes for subsequent studies.Conclusion:Peptides derived from Pol and X proteins have been successfully isolated and identified,and potential T cell epitopes have been screened.

AIM:To study the improvement effect and possible mechanism of Shufeng Jiedu capsules(SFJDC)on viral pneumonia.METHODS:Forty-eight BALB/c mice were used to establish a viral pneumo-nia model by intranasal administration of influenza A virus H1N1.They were randomly divided into 4 groups,with twelve mice in each group.Except for the Model group,the treatment group was given oseltamivir(OSTW),low and high dose groups of Shufeng Jiedu capsules(SFJDC-L,SFJD-H)by gavage,take another 12 mice as the normal group,continu-ously administered for 7 days.The physical signs of each group of mice were observed during the ad-ministration process,and the daily body weight change rate and disease activity index was calculat-ed;After administration,lung,intestinal tissue,and intestinal contents were taken,and the pathological changes in mouse lung tissue were detected using hematoxylin eosin(HE)staining method,Alisin blue staining was used to detect goblet cells in the intes-tinal wall;RT-qPCR method for detecting viral load influenza(infA)and the expression of tumor necro-sis factor-a(TNF-α)and interleukin-6(IL-6)mRNA in mouse lung tissue;Detection of mouse gut mi-crobiota using 16S rRNA high-throughput sequenc-ing;ELISA method for detecting lipopolysaccharide(LPS),TNF-α and IL-6 level in mouse serum;The ex-pression of cyclic GMP AMP synthase(cGAS),inter-feron gene stimulating protein(STING),phospho-STING(p-STING),nuclear factor-κB(NF-κB)phos-pho-nuclear factor-κB(p-NF-κB)in lung tissue and ZO-1,Occludin in small small intestine was detected by Western Blotting.RESULTS:Compared with the model group,the lung index and viral load of mice in the Shufeng Jiedu capsules group were signifi-cantly reduced(P＜0.01);significant reduction in lung tissue inflammation accompanied by TNF-α;The expression level of IL-6 mRNA decreased(P＜0.01);The abundance of Firmicutes and Lachnospi-raceae in the intestine significantly increased(P＜0.01),while the abundance of Bacteroidetes and Bacteroidaceae communities significantly de-creased(P＜0.05 or 0.01);The number of goblet cells in the small intestine epithelium significantly increased(P＜0.01);the levels of serum LPS and TNF-α,IL-6 were significantly reduced(P＜0.01);The protein expression level of cGAS,p-STING,p-NF-κB significantly decreased in lung tissue(P＜0.05 or 0.01),ZO-1,Occludin significantly decreased in small intestine(P＜0.05).CONCLUSION:Shufeng Jie-du capsules reduces the inflammatory response of mice lung tissue,and its mechanism may be relat-ed to its regulation of intestinal flora,protection of intestinal mucosal barrier,and reduction the activa-tion of cGAS/STING/NF-κB signaling pathway ac-tived by LPS leakaging from the intestine.

AIM:To study the improvement effect and possible mechanism of Shufeng Jiedu capsules(SFJDC)on viral pneumonia.METHODS:Forty-eight BALB/c mice were used to establish a viral pneumo-nia model by intranasal administration of influenza A virus H1N1.They were randomly divided into 4 groups,with twelve mice in each group.Except for the Model group,the treatment group was given oseltamivir(OSTW),low and high dose groups of Shufeng Jiedu capsules(SFJDC-L,SFJD-H)by gavage,take another 12 mice as the normal group,continu-ously administered for 7 days.The physical signs of each group of mice were observed during the ad-ministration process,and the daily body weight change rate and disease activity index was calculat-ed;After administration,lung,intestinal tissue,and intestinal contents were taken,and the pathological changes in mouse lung tissue were detected using hematoxylin eosin(HE)staining method,Alisin blue staining was used to detect goblet cells in the intes-tinal wall;RT-qPCR method for detecting viral load influenza(infA)and the expression of tumor necro-sis factor-a(TNF-α)and interleukin-6(IL-6)mRNA in mouse lung tissue;Detection of mouse gut mi-crobiota using 16S rRNA high-throughput sequenc-ing;ELISA method for detecting lipopolysaccharide(LPS),TNF-α and IL-6 level in mouse serum;The ex-pression of cyclic GMP AMP synthase(cGAS),inter-feron gene stimulating protein(STING),phospho-STING(p-STING),nuclear factor-κB(NF-κB)phos-pho-nuclear factor-κB(p-NF-κB)in lung tissue and ZO-1,Occludin in small small intestine was detected by Western Blotting.RESULTS:Compared with the model group,the lung index and viral load of mice in the Shufeng Jiedu capsules group were signifi-cantly reduced(P＜0.01);significant reduction in lung tissue inflammation accompanied by TNF-α;The expression level of IL-6 mRNA decreased(P＜0.01);The abundance of Firmicutes and Lachnospi-raceae in the intestine significantly increased(P＜0.01),while the abundance of Bacteroidetes and Bacteroidaceae communities significantly de-creased(P＜0.05 or 0.01);The number of goblet cells in the small intestine epithelium significantly increased(P＜0.01);the levels of serum LPS and TNF-α,IL-6 were significantly reduced(P＜0.01);The protein expression level of cGAS,p-STING,p-NF-κB significantly decreased in lung tissue(P＜0.05 or 0.01),ZO-1,Occludin significantly decreased in small intestine(P＜0.05).CONCLUSION:Shufeng Jie-du capsules reduces the inflammatory response of mice lung tissue,and its mechanism may be relat-ed to its regulation of intestinal flora,protection of intestinal mucosal barrier,and reduction the activa-tion of cGAS/STING/NF-κB signaling pathway ac-tived by LPS leakaging from the intestine.

Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-class Ⅰ complex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-class Ⅰ and Ⅱ molecules and for prediction of the binding ability of the peptide HLA-class Ⅰ complexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-class Ⅰ and Ⅱ molecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC50<500 nmol/L to HLA-class Ⅰ and Ⅱ molecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding geno-types.Peptides with affinity IC50<500 nmol/L had an affinity core sequence overlap of no less than 8 amino acids,and can be priori-tized for candidate T cell epitopes for subsequent studies.Conclusion:Peptides derived from Pol and X proteins have been successfully isolated and identified,and potential T cell epitopes have been screened.

BACKGROUND:As tissue engineering brings new hope to the worldwide problem of articular cartilage repair,the construction of light-curing 3D printed hydrogel scaffolds with biomimetic composition is of great significance for cartilage tissue engineering. OBJECTIVE:To construct a biomimetic methacryloylated hyaluronic acid/acellular Wharton's jelly composite hydrogel scaffold by digital light processing 3D printing technology,and to evaluate its biocompatibility. METHODS:Wharton's jelly was isolated and extracted from human umbilical cord,then decellulated,freeze-dried,ground into powder,and dissolved in PBS to prepare 50 g/L acellular Wharton's jelly solution.Methylallylated hyaluronic acid was prepared,lyophilized and dissolved in PBS to prepare 50 g/L methylallylated hyaluronic acid solution.Acellular Wharton's jelly solution was mixed with methacrylyacylated hyaluronic acid solution at a volume ratio of 1:1,and was used as bio-ink after adding photoinitiator.Methylacrylylated hyaluronic acid hydrogel scaffolds(labeled as HAMA hydrogel scaffolds)and methylacrylylated hyaluronic acid/acellular Wharton's jelly gel scaffolds(labeled as HAMA/WJ hydrogel scaffolds)were prepared by digital light processing 3D printing technology,and the microstructure,swelling performance,biocompatibility,and cartilage differentiation performance of the scaffolds were characterized. RESULTS AND CONCLUSION:(1)Under scanning electron microscope,the two groups of scaffolds showed a three-dimensional network structure,and the fiber connection of HAMA/WJ hydrogel scaffold was more uniform.Both groups achieved swelling equilibrium within 10 hours,and the equilibrium swelling ratio of HAMA/WJ hydrogel scaffold was lower than that of HAMA hydrogel scaffold(P＜0.05).(2)CCK-8 assay showed that HAMA/WJ hydrogel scaffold could promote the proliferation of bone marrow mesenchymal stem cells compared with HAMA hydrogel scaffold.Dead/live staining showed that bone marrow mesenchymal stem cells grew well on the two groups of scaffolds,and the cells on the HAMA/WJ hydrogel scaffolds were evenly distributed and more cells were found.Phalloidine staining showed better adhesion and spread of bone marrow mesenchymal stem cells in HAMA/WJ hydrogel scaffold than in HAMA.(3)Bone marrow mesenchymal stem cells were inoculated into the two groups for chondrogenic induction culture.The results of qRT-PCR showed that the mRNA expressions of agglutinoglycan,SOX9 and type Ⅱ collagen in the HAMA/WJ hydrogel scaffold group were higher than those in the HAMA hydrogel scaffold group(P＜0.05,P＜0.01).(4)These findings indicate that the digital light processing 3D bioprinting HAMA/WJ hydrogel scaffold can promote the proliferation,adhesion,and chondrogenic differentiation of bone marrow mesenchymal stem cells.

Articular cartilage (AC) injuries often lead to cartilage degeneration and may ultimately result in osteoarthritis (OA) due to the limited self-repair ability. To date, numerous intra-articular delivery systems carrying various therapeutic agents have been developed to improve therapeutic localization and retention, optimize controlled drug release profiles and target different pathological processes. Due to the complex and multifactorial characteristics of cartilage injury pathology and heterogeneity of the cartilage structure deposited within a dense matrix, delivery systems loaded with a single therapeutic agent are hindered from reaching multiple targets in a spatiotemporal matched manner and thus fail to mimic the natural processes of biosynthesis, compromising the goal of full cartilage regeneration. Emerging evidence highlights the importance of sequential delivery strategies targeting multiple pathological processes. In this review, we first summarize the current status and progress achieved in single-drug delivery strategies for the treatment of AC diseases. Subsequently, we focus mainly on advances in multiple drug delivery applications, including sequential release formulations targeting various pathological processes, synergistic targeting of the same pathological process, the spatial distribution in multiple tissues, and heterogeneous regeneration. We hope that this review will inspire the rational design of intra-articular drug delivery systems (DDSs) in the future.

Objective:To understand the risk signal of ocular adverse events (AE) related to mycophenolate mofetil (MMF) and to provide reference for the safe clinical use of this drug.Methods:The US FDA Adverse Event Reporting System database was searched, and the AE reports on MMF as the primary suspect drug from the 1st quarter of 2004 to the 3rd quarter of 2022 were collected. AEs were counted and classified using the preferred system organ class (SOC) and preferred term (PT) of Medical Dictionary for Regulatory Activities version 24.0, and ocular AEs were screened out. The ocular AE risk signals were explored using 3 frequency methods, including reporting odds ratio (ROR) method, proportional reporting ratio (PRR) method, and Bayesian confidence propagation neural network method, and the multi-item gamma-Possion shrinker (MGPS) method. The information of the ocular AE reports and AE risk signals of MMF were analyzed descriptively. Results:A total of 402 cases of ocular AE with MMF as the primary suspect drug were collected, which involved 402 patients, 31 PTs and 5 SOCs. The 402 AE cases were reported among 33 countries, 283 of which had clinical outcome records, including death in 32 cases (11.3%), disability or blindness in 142 cases (50.2%), life-threatening in 14 cases (4.9%), and hospitalization or prolonged hospitalization in 95 cases (33.6%). Results of the frequency method showed that all 31 PTs were risk signals, while the results of the MGPS method manifested that 22 PTs were risk signals. None of the 31 PTs were recorded in the drug labels. The top 5 PTs in the number of AE reports were blindness (136 cases), cytomegalovirus chorioretinitis (37 cases), uveitis (34 cases), endophthalmitis (29 cases), and necrotising retinitis (22 cases). The ranking of signal intensity showed by the 4 methods was similar. The top 5 PTs with the high signal intensity were orbital apex syndrome [ ROR=55.84, PRR=55.83, information component ( IC)=5.58, empirical Bayesian geometric mean ( EBGM)=47.71], quadrantanopia ( ROR=43.22, PRR=43.21, IC=5.26, EBGM=38.21), retinitis viral ( ROR=40.13, PRR=40.13, IC=5.16, EBGM=35.78), optic discs blurred ( ROR=40.13, PRR=40.13, IC=5.16, EBGM=35.78), and serpiginous choroiditis ( ROR=31.07, PRR=31.07, IC=4.83, EBGM=28.41). Conclusions:The clinical manifestations of ocular AE during MMF treatment are diverse, and none of them are recorded in the drug label. The clinical outcomes are poor and can lead to blindness, which should be vigilant in clinical practice.

Objective:To understand the risk signal of ocular adverse events (AE) related to mycophenolate mofetil (MMF) and to provide reference for the safe clinical use of this drug.Methods:The US FDA Adverse Event Reporting System database was searched, and the AE reports on MMF as the primary suspect drug from the 1st quarter of 2004 to the 3rd quarter of 2022 were collected. AEs were counted and classified using the preferred system organ class (SOC) and preferred term (PT) of Medical Dictionary for Regulatory Activities version 24.0, and ocular AEs were screened out. The ocular AE risk signals were explored using 3 frequency methods, including reporting odds ratio (ROR) method, proportional reporting ratio (PRR) method, and Bayesian confidence propagation neural network method, and the multi-item gamma-Possion shrinker (MGPS) method. The information of the ocular AE reports and AE risk signals of MMF were analyzed descriptively. Results:A total of 402 cases of ocular AE with MMF as the primary suspect drug were collected, which involved 402 patients, 31 PTs and 5 SOCs. The 402 AE cases were reported among 33 countries, 283 of which had clinical outcome records, including death in 32 cases (11.3%), disability or blindness in 142 cases (50.2%), life-threatening in 14 cases (4.9%), and hospitalization or prolonged hospitalization in 95 cases (33.6%). Results of the frequency method showed that all 31 PTs were risk signals, while the results of the MGPS method manifested that 22 PTs were risk signals. None of the 31 PTs were recorded in the drug labels. The top 5 PTs in the number of AE reports were blindness (136 cases), cytomegalovirus chorioretinitis (37 cases), uveitis (34 cases), endophthalmitis (29 cases), and necrotising retinitis (22 cases). The ranking of signal intensity showed by the 4 methods was similar. The top 5 PTs with the high signal intensity were orbital apex syndrome [ ROR=55.84, PRR=55.83, information component ( IC)=5.58, empirical Bayesian geometric mean ( EBGM)=47.71], quadrantanopia ( ROR=43.22, PRR=43.21, IC=5.26, EBGM=38.21), retinitis viral ( ROR=40.13, PRR=40.13, IC=5.16, EBGM=35.78), optic discs blurred ( ROR=40.13, PRR=40.13, IC=5.16, EBGM=35.78), and serpiginous choroiditis ( ROR=31.07, PRR=31.07, IC=4.83, EBGM=28.41). Conclusions:The clinical manifestations of ocular AE during MMF treatment are diverse, and none of them are recorded in the drug label. The clinical outcomes are poor and can lead to blindness, which should be vigilant in clinical practice.

With the emergence of DNA nanotechnology in the 1980s, self-assembled DNA nanostructures have attracted considerable attention worldwide due to their inherent biocompatibility, unsurpassed programmability, and versatile functions. Especially promising nanostructures are tetrahedral framework nucleic acids (tFNAs), first proposed by Turberfield with the use of a one-step annealing approach. Benefiting from their various merits, such as simple synthesis, high reproducibility, structural stability, cellular internalization, tissue permeability, and editable functionality, tFNAs have been widely applied in the biomedical field as three-dimensional DNA nanomaterials. Surprisingly, tFNAs exhibit positive effects on cellular biological behaviors and tissue regeneration, which may be used to treat inflammatory and degenerative diseases. According to their intended application and carrying capacity, tFNAs could carry functional nucleic acids or therapeutic molecules through extended sequences, sticky-end hybridization, intercalation, and encapsulation based on the Watson and Crick principle. Additionally, dynamic tFNAs also have potential applications in controlled and targeted therapies. This review summarized the latest progress in pure/modified/dynamic tFNAs and demonstrated their regenerative medicine applications. These applications include promoting the regeneration of the bone, cartilage, nerve, skin, vasculature, or muscle and treating diseases such as bone defects, neurological disorders, joint-related inflammatory diseases, periodontitis, and immune diseases.
Nucleic Acids/chemistry* ; Regenerative Medicine ; Consensus ; Reproducibility of Results ; DNA/chemistry*

Due to good mechanical properties and biocompatibility, tissue engineering scaffolds have become the vital method for repairing and regenerating articular cartilage defects. With the continuous development of tissue engineering technology, many scaffolds preparation and formation methods have been developed and tested in the past decade, however, the preparation of ideal regenerative scaffolds remain controversial. As load-bearing tissue inside the body joints, the matrix structure and cell composition of articular cartilage are hierarchical, and there are several smooth natural gradients from the cartilage surface to the subchondral bone layer, including cell phenotype and number, specific growth factors, matrix composition, fiber arrangement, mechanical properties, nutrient and oxygen consumption. Therefore, in the design of regenerative scaffolds, it is necessary to achieve these gradients to regenerate articular cartilage in situ. In recent studies, many new biomimetic gradient scaffolds have been used to simulate the natural gradient of articular cartilage. These scaffolds show different mechanical, physicochemical or biological gradients in the structure, and have achieved good repair effects. The related articles on tissue engineering for the treatment of articular cartilage defects were retrieved by searching databases with key wordsarticular cartilage injury, cartilage repair and gradient scaffolds. In this work,the structural, biochemical, biomechanical and nutrient metabolism gradients of natural articular cartilage were studied and summarized firstly. Then, the latest design and construction of articular cartilage gradient scaffolds were classified. Besides that, the material composition (such as hydrogels, nanomaterials, etc.) and the preparation process (such as electrospinning, 3D printing, etc.) of grandient scaffolds were further enhanced. Finally, the prospect and challenge of biomimetic gradient scaffolds in cartilage engineering are discussed, which provides a theoretical basis for the successful application of gradient scaffolds in clinical transformation.