Preparation,identification and comparison of murine and rabbit polyclonal antibodies against matrix protein of rabies virus strain CVS-11 / 中国生物制品学杂志

@#Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV) matrix(M) protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA) and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1:100 and 1:256 000respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.

LED red light up-regulates MAPK signal to promote osteogenic/odontogenic differentiation of human dental pulp stem cells in an inflammatory environment / 口腔疾病防治

Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.

Changes of immune microenvironment and prognosis of bladder cancer patients with positive urinary nuclear matrix protein 22 / 中国医师杂志

Objective:To explore the changes of immune microenvironment and prognosis of bladder cancer patients with positive urinary nuclear matrix protein 22 (NMP22).Methods:Retrospective analysis was made on 86 patients who were diagnosed with bladder cancer in Xuzhou Central Hospital from January 2019 to September 2020. All patients were tested for urinary NMP22 by colloidal gold method. The patients with positive test results were NMP22 positive group, and the patients with negative test results were NMP22 negative group. The expression of CD8, programmed cell death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1) and PanCK were detected by multiple fluorescent immunohistochemical method on the pathological tissue sections of all enrolled patients with bladder cancer after surgery. Follow-up data of enrolled patients were collected after discharge, and univariate and multivariate Cox analysis was performed on the follow-up data.Results:There were 69 patients in the NMP22 positive group and 17 patients in the NMP22 negative group. The percentage of CD8 and PD-L1 positive cells in NMP22 positive group was significantly higher than that in NMP22 negative group, and the difference was statistically significant (all P<0.05). Univariate analysis showed that tumor stage was correlated with bladder cancer progression ( HR=2.67, P=0.017). Multivariate analysis showed that positive NMP22 was significantly correlated with bladder cancer recurrence and disease progression (all P<0.05). Conclusions:The density of CD8 + T cells and PD-L1 in tumor parenchyma of urinary NMP22 positive bladder cancer patients was higher than that of NMP22 negative patients. Urinary NMP22 positive can be one of the bad prognostic factors of bladder cancer, and the patients with NMP22 positive should strengthen reexamination.

Study on the relationship between local muscle fatigue and related metabolic biomarkers in simulated manual lifting / 中国职业医学

{L-End}Objective To analyze the local muscle response under continuous ergonomic workload by simulating manual lifting, and to screen the sensitive metabolic biomarkers during fatigue process. {L-End}Methods A total of 13 healthy male volunteers were selected as the study subjects using simple random sampling method. Study subjects underwent repetitive simulated manual lifting for four periods (T1 to T4), each lasting 12 minutes. The degree of work-related fatigue in the forearm, upper arm, shoulder, back, and leg muscles, and the whole body was accessed using Borg 6-20 Rating of Perceived Exertion (RPE) Scale. The venous blood samples were collected from elbow between each two periods to detect the levels of eight metabolic biomarkers: ammonia, lactate, creatine kinase, lactate dehydrogenase (LDH), C-terminal telopeptide of type Ⅰ collagen (CTX-Ⅰ), C-terminal telopeptide of type Ⅱ collagen (CTX-Ⅱ), cartilage oligomeric matrix protein (COMP), and calcium ions. {L-End}Results The RPE scores of the study subjects for the muscles of five body parts and the whole body increased with the increasing lifting periods (all P<0.01). Fatigue was observed in all target muscles, with overall body fatigue occurring in the T2 period. The levels of ammonia, lactate, creatine kinase, LDH, COMP, and calcium ions in the serum of study subjects were higher in the T1 to T4 periods than in the T0 period (all P<0.05). The serum CTX-Ⅰ level was higher in the T1 and T3 periods than that in the T0 period (all P<0.05) , and the serum CTX-Ⅱ level was higher in the T1, T2 and T4 periods than that in the T0 period (all P<0.05). The level of these eight serum metabolic biomarkers fluctuated during the T1 to T4 periods. The serum creatine kinase level increased with the period of lifting (all P<0.05). The serum lactate level was higher in the T3 period than those in the T1 and T2 periods (all P<0.05). The serum LDH and calcium ion levels were higher in the T2 to T4 periods than that in the T1 period (all P<0.05). The serum COMP level was higher in the T2 and T3 periods than that in the T1 period (all P<0.05). Except for CTX-Ⅰ, the levels of other seven metabolic markers in serum were higher in individuals after fatigue than before fatigue (all P<0.05). {L-End}Conclusion Serum metabolic biomarkers such as ammonia, lactate, creatine kinase, calcium ions, LDH, CTX-Ⅱ, and COMP exhibit significant changes before and after fatigue. These metabolic biomarkers could be used as sensitive biomarkers for evaluating muscle fatigue during repetitive works.

Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSVMT) in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.

Immune responses elicited by influenza A mRNA vaccine based on lipopolyplex-encapsulated virus nucleoprotein and matrix protein 2 extracellular domain fusion in mice / 中华微生物学和免疫学杂志

Objective:To evaluate the immunogenicity of a novel influenza virus mRNA vaccine based on conserved antigens delivered by lipopolyplex (LPP) platform in a mouse model.Methods:Four copies of genes coding for extracellular domain of matrix 2 protein (M2e) and nucleoprotein (NP) of influenza A virus were synthetized after codon optimization. The fusion antigens were transcribed in vitro and delivered by LPP platform, named as LPP-4M2eNP. Expression of M2e and NP in eukaryotic cells was detected by immunofluorescence assay (IFA). BALB/c mice were inoculated intramuscularly twice with 10 μg or 30 μg LPP-4M2eNP vaccine at an interval of four weeks. Antibody response was detected by ELISA and cellular-mediated immunity (CMI) was detected by enzyme-linked immunospot assay (ELISPOT). Results:IFA showed that NP and M2e were expressed correctly in eukaryotic cells. Single dose immunization could induce significant antigen (NP, M2e)-specific CMI and antigen (NP, M2e)-specific antibody response was induced in mice with Th1 type bias after boost immunization. Moreover, NP-specific CMI was increased significantly after the second immunization, while no significant change in M2e-specific CMI was observed.Conclusions:Stronger CMI was triggered in mice by single dose of LPP-4M2eNP vaccine. Furthermore, robust humoral and cellular immune responses were induced after boost immunization. This study suggested that LPP-4M2eNP vaccine, which based on conserved antigen of influenza A and delivered by LPP platform, had great potential for development and application.

Correlation between proteoglycan and alveolar bone resorption in a mouse periodontitis model / 口腔疾病防治

Objective@#To analyze changes in proteoglycan and its correlation with alveolar bone resorption in periodontitis. @*Methods @#Twelve eight-week-old C57BL/6J male mice were selected, and the periodontitis model was established by ligating the right maxillary second molar with 6-0 silk thread. The nonligated part of the left maxilla was used as the control. The mice were killed 14 days after the operation. Micro-CT was used to assess alveolar bone resorption. HE staining was used to observe the alveolar bone profile, and TRAP staining was conducted to examine the positive rate of osteoclasts. The expression of proteoglycan-related genes, such as aggrecan (ACAN), biglycan (BGN), versican (VCAN), decorin (DCN), osteoclast-related genes, such as cathepsin K (CTSK), matrix metalloprotein-9 (MMP-9), and receptor activator of nuclear factor kappa-B ligand (RANKL), and inflammation-related genes, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), was detected by real-time quantitative PCR. Additionally, the correlation of the expression of proteoglycans with osteoclast-related genes and inflammation-related genes was evaluated by Pearson correlation analysis.@* Results@#The resorption of alveolar bone on the periodontitis side increased. TRAP staining showed that the number of osteoclasts was substantially increased in the maxilla with periodontitis. Real-time quantitative PCR demonstrated that compared with the control side, the expression of proteoglycan-related genes, such as ACAN, BGN, and DCN, was decreased, whereas the expression of the VCAN gene was significantly increased in the periodontitis side. Meanwhile, the expression of osteoclast-related genes, such as CTSK, MMP-9, and RANKL, and inflammation-related genes, such as IL-1β, IL-6, and TNF-α, was markedly increased in the periodontitis side (P<0.05). Pearson correlation analysis indicated a negative correlation between the expression of proteoglycans and the mRNA levels of osteoclast-related genes and inflammation-related genes (P<0.05). @*Conclusion @#The expression of proteoglycan was closely related to alveolar bone resorption in a periodontitis model.

The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

Autosomal recessive hypophosphatemic rickets 1 caused by DMP1 mutation: one pedigree study / 中华内分泌代谢杂志

In the present study, the clinical features of a patient with autosomal recessive hypophosphatemic rickets 1 caused by dentin matrix protein 1(DMP1)gene mutation and her family members were investigated. DMP1 gene from peripheral blood was sequenced by Sanger sequencing, and the known mutation was verified among her family members and 250 healthy populations. The proband was a 42-year-old female with bone deformity of both lower limbs, bone pain, and short stature. The results of X-rays and laboratory examination were consistent with the hypophosphatemic rickets reported before. A homozygous mutation(c.2T> C)in DMP1 was identified by Sanger sequencing in the proband, her son and daughter were heterozygous for c. 2T> C.

Diagnostic Accuracy of Cartilage Oligomeric Matrix Protein (COMP), for Cartilage Damage in Rheumatoid Arthritis

Background:Cartilage oligomeric matrix protein (COMP), is an extracellular matrix (ECM) non-collagenous glycoprotein that is mainly localized within the cartilage, and also be found in tendon and synovium.RecentstudiesinwestandAsiaPacificregionhasshownthatCOMP, is a prognostic marker in Rheumatoid arthritis(RA).Objective:To correlate serum COMP levels with disease severity and cartilage destruction in rheumatoid arthritis.Methods:The study was conducted in Department of Pathology and Rheumatology, Ziauddin University Hospital, Karachi from June 2018 to May 2019. Patients were recruited as per American College of Rheumatology (ACR) 2010 classification criteria. The study populationconsists of 88 healthy subjects and 88 RA patients. Sandwich ELISA technique was used to assess serum COMP level. Other inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) antibodies like rheumatoid factor, and anti-cyclic citrullinated protein (anti-CCP) were also assessed. Results were analyzed using SPSS-20 and P-value ≤0.05 was considered as significant.Results: Serum COMP levels were significantly higher in RA patients 51.35ng/ml than controls 21.454ng/ml with significant p value=<0.0001. There was strong positive correlation between COMP level and disease severity in RA patients with moderate as well as high disease activity score (DAS) with significant p value. Serum COMP showed 96% sensitivity and 83% specificity at level of 27.01ng/ml for diagnosis of RA.Conclusions:COMP has significant positive correlation with severity of RA. Serum COMP can be utilized as a biomarker to quantify cartilage destruction in RA patients

Extension and dentin differentiation potential of dental pulp stem cells from human deciduous teeth on the stiff matrix surface / 中国组织工程研究

BACKGROUND: Dental pulp stem cells can differentiate into dentin under appropriate induction conditions, which are important seed cells i n dental tissue engineering. However, the commonly used inducers are chemical agents, which are not available for in vivo application. Mesenchymal stem cells can differentiate with the material hardness, and the physical property-induced cell differentiation is little reported. OBJECTIVE: To observe the extension characteristics and dentin differentiation potential of dental pulp stem cells from human deciduous teeth on the stiff matrix surface. METHODS: Dental pulp stem cells from naturally shed deciduous teeth were isolated, cultured and identified. Four solid gel matrixes with elasticity modulus of (9.12±0.94), (27.18±3.55), (59.37±4.05) and (86.45±5.33) kPa were made using low melting point agarose. The extension ability of passage 4 dental pulp stem cells on the surface of the above solid matrixes was detected by two-dimensional clone formation and cell scratch tests. The protein expression levels of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein were detected by western blot assay. RESULTS AND CONCLUSION: Dental pulp stem cells from human deciduous teeth seeded on the gel matrix with extremely low and low hardness almost existed as cell clones with neat edges, and cell spreading and extension were rare. When seeded on the gel matrix with moderate and high hardness, the cloned edge of deciduous dental pulp stem cells spread and extended obviously. The cell body became large and the cell edge extended significantly. The cell scratch test revealed the similar phenomenon. When seeded on the gel matrix with moderate and high hardness, dental pulp stem cells from human deciduous teeth exhibited high expression levels of of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein. In summary, with the increase of matrix hardness, the abilities of extension and differentiation into dentin of dental pulp stem cells from human deciduous teeth are increased gradually, which provides a method for dental tissue engineering.

Effects and mechanisms of sodium ibandronate in ovariectomy-induced osteoporosis rats / 中国组织工程研究

BACKGROUND: Bisphosphonates are a novel inhibitor of bone resorption that can inhibit the activity and function of osteoclasts. OBJECTIVE: To observe the effects of sodium ibandronate on the expression of dentin matrix protein 1, Caspace3, Bcl-2 and Bax in condylar cartilage in osteoporosis rats. METHODS: Thirty-six female rats were randomly divided into sham group, osteoporosis group and sodium ibandronate group, twelve in each group. The sham group did not excise ovaries during surgery. Bilateral ovaries of rats were removed in the osteoporosis and sodium ibandronate groups. On the 7th day after operation, rats in the sodium ibandronate group were intraperitoneally given sodium ibandronate 10 µg/kg, once every 7 days. After 90 days, the rat ovaries were taken. Bone mineral density was measured in each group. The changes of condylar cartilage were observed by toluidine blue staining and TUNEL staining. The expression of dentin matrix protein 1 protein was detected by immunohistochemistry. The levels of Caspase3, Bcl-2 and Bax were detected by western blot assay. The study protocol was approved by the Animal Ethics Committee of Nanhua Hospital in China with the approval No. SLXD_201804010. RESULTS AND CONCLUSION: Compared with the sham group or sodium ibandronate group, the bone mineral density in the osteoporosis group was significantly decreased (P < 0.05). The results of toluidine blue staining showed that the hypertrophic layer of condylar cartilage in the sodium ibandronate group was significantly thicker than that in the osteoporosis group. Compared with the sham group or sodium ibandronate group, the number of apoptotic cells in condylar cartilage and subchondral bone increased significantly in the osteoporosis group (P < 0.05). The expression of dentin matrix protein 1 protein was significantly lower in the osteoporosis group than the sham group, but it increased after treatment with sodium ibandronate (P < 0.05). Compared with the sham group, the expression of Caspase 3 and Bax in the osteoporosis group increased significantly, and the expression of Bcl-2 decreased. However, treatment with sodium ibandronate decreased the expression of Caspase 3 and Bax and increased the expression of Bcl-2 significantly. Overall, our findings reveal that sodium ibandronate can inhibit the apoptosis of condylar chondrocytes and the number of osteoclasts in osteoporotic state, which may be related to the regulation of Caspase 3, Bcl-2, Bax and dentin matrix protein 1 expression.

Expression of cartilage oligomeric matrix protein in the synovial chondromatosis of the temporomandibular joint / 北京大学学报(医学版)

OBJECTIVE@#To detect the expression of cartilage oligomeric matrix protein (COMP) in the synovial chondromatosis of the temporomandibular joint (TMJSC), and to discuss the possible interactions between COMP, transforming growth factor (TGF)-β3, TGF-β1 and bone morphogenetic protein-2 (BMP-2) in the development of this neoplastic disease.@*METHODS@#Patients in Peking University School and Hospital of Stomatology from January 2011 to February 2020 were selected, who had complete medical records, TMJSC was verified histologically after operation. The expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in the TMJSC of the temporomandibular joint were detected by immunohistochemistry and quantitative real-time PCR (RT-PCR) at the protein level and mRNA level respectively, compared with the normal synovial tissue of temporomandibular joint. The histological morphology, protein expression and distribution of TMJSC tissues were observed microscopically, and the positive staining proteins were counted and scored. SPSS 22.0 statistical software was used to analyze the expression differences between the related proteins in TMJSC tissue and the normal synovial tissue of temporomandibular joint and to compare their differences. P < 0.05 indicated that the difference was statistically significant.@*RESULTS@#Immunohistochemical results showed that the positive expression of COMP in TMJSC tissues was mostly found in synovial tissues and chondrocytes adjacent to synovial tissues, and the difference was statistically significant, compared with the normal temporomandibular joint synovial tissues. The positive expression of COMP was significantly different between recurrent TMJSC and non-recurrent ones. The positive expressions of TGF-β3, TGF-β1 and BMP-2 were higher than the normal synovial tissue, and were also mostly found in the synovial cells and adjacent chondrocytes, which was further confirmed by Western blot. According to the RT-PCR results, the expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in TMJSC were higher than those in the normal synovial tissue.@*CONCLUSION@#The expression of COMP in TMJSC of temporomandibular joint increased significantly, compared with the normal synovial tissue. There may be interactions between COMP and cytokines related to the proliferation and differentiation, like TGF-β3, TGF-β1 and BMP-2, which may play a potential role in the pathogenesis of TMJSC.

Effects of Aconitum Injection on COMP ，p53 Protein and BMP- 2 in Knee Osteoarthritis Model Rabbits / 中国药房

OBJECTIVE：To investigate the effects of Aconitum injection on cartilage oligomeric matrix protein （COMP）， encoded protein by tumor suppressor gene p53（p53 protein）and bone morphogenetic protein 2（BMP-2）in knee osteoarthritis （KOA）model rabbit ，so as to explore the mechanism of the injection in the treatment of KOA. METHODS ：Totally 24 rabbits were randomly divided into blank group ，model group ，Sodium hyaluronate group and Aconitum injection group ，with 6 rabbits in each group. KOA model was established by injecting 2％ papain-0.03 mol/L L-cysteine solution into the articular cavity of rabbits in model group ，sodium hyaluronate group and Aconitum injection group at the 1st，4th and 7th day ，respectively，except the blank group. At the 1st，4th and 7th day after modeling succeeded ，0.1 mL/kg of normal saline ，Sodium hyaluronate injection and Aconitum injection were injected into the articular cavity of rabbits ，respectively. The cartilage tissue of knee joint was taken from above 4 groups，and the contents of COMP and p 53 protein were detected by ELISA. The cartilage morphology of rabbit knee joint was observed by naked eye. The cartilage of the knee joint was collected and stained by HE staining ，and then the histomorphology changes were observed by light microscope ；Mankin scoring was conducted. The two-step method of PV was used to make the immunohistochemical specimens of knee joint cartilage ，and the relative expression of BMP- 2 was detected. RESULTS ：Compared with blank group ，the edge of cartilage was damaged and the cartilage surface was damaged in the model group. The results of histomorphology observation showed that the joint tissue structure was obviously irregular ，the distribution of chondrocytes was disordered with morphological changes ，and the Mankin score was significantly increased （P＜0.05）；the contents of COMP and cancer cells by indirect inhibition of RAD 51-mediated re - . Suppression of ERCC 1 and RAD51 expression through ERK 1/2 inactivation is essen - tial in emodin-mediated cytotoxicity in human non-small 。E-mail： cell lung cancer cells. p53 protein in knee joint fluid were increased significantly ，while the relative expression of BMP- 2 in knee joint tissue decreased significantly （P＜0.05）. Compared with model group ，the appearance ，histomorphology changes of knee joint cartilage in administration groups were improved，Mankin scores were significantly decreased （P＜0.05）；the contents of COMP and p 53 protein in the knee joint fluid were decreased significantly ，and the relative expression of BMP- 2 in the knee joint tissue were increased significantly （P＜0.05），but there was no significant difference between Aconitum group and Sodium hyaluronate group （P＞0.05）. CONCLUSIONS ：Aconitum injection can improve synovitis inflammation ，delay articular cartilage degeneration ， promote cartilage repair and protect joints of KOA model rabbits. The mechanism may be related to inhibiting COMP secretion ， decreasing p 53 protein expression and promoting BMP- 2 release.

Advances in the research of extracellular matrix protein prediction tools / 生物工程学报

Extracellular matrix (ECM) proteins play an important role in a series of biological processes in the cell, and their abnormal regulation can lead to many diseases. The theoretical ECM reference dataset is the basis for efficient identification of extracellular matrix proteins. Researchers have developed various ECM protein prediction tools based on machine learning methods. In this review, the main strategy of development of ECM protein prediction tools that based on machine learning methods has been introduced. Then, advances and specific characters of the existing ECM protein prediction tools have been summarized. Finally, the challenges and possible improvements of ECM protein prediction tools are discussed.

Enhance the affinity of nanobodies to CD47 antigen by fusion expression of COMP48 self-folding peptide / 国际生物医学工程杂志

Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90％homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant ( P<0 . 01 ) . Through non-competitive ELISA , the constants of affinity and dissociation constants were 6.97 ×107 L/mol and 1.434 ×10-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody.

Clinical characteristics of pseudoachondroplasia and analysis of COMP gene mutation / 中华内分泌代谢杂志

Objective@#This article reported the clinical characteristics and gene mutations of two pseudoachondroplasia cases, and made a literature review in order to improve clinicians′ understanding of the disease.@*Methods@#Clinical features of two patients who were short stature accompanied with skeletal deformities were summarized, and they accepted whole exome sequencing. We also reviewed literature to summarize the clinical characteristics and known gene research progress of all reported Chinese pseudoachondroplasia cases.@*Results@#The two patients′ clinical characteristics were short limbdwarfism with skeletal deformity. Genetic results showed that there were two heterozygous mutations in the cartilage oligomeric matrix protein (COMP) gene of the two patients, c. 1417_1419delGAC and c. 1552G>A, respectively. Up to March 2019, a total of 58 cases of pseudoachondroplasia have been reported in China. The median height of these patients is -5.03 SDS. The clinical features include abnormal gait, short limbs, short fingers/toes, scoliosis, bracelet sign, ankle sign and other skeletal deformities. COMP is the pathogenic gene and mutations mainly located in calmodulin-like domains. The hotspot mutation is c. 1417_1419delGAC.@*Conclusions@#Pseudoachondroplasia is a kind of rare genetic disease characterized by short stature and skeletal deformities. The clinical and genetic characteristics of the disease were summarized, which may improve the early diagnosis rate.

Clinical characteristics of pseudoachondroplasia and analysis of COMP gene mutation / 中华内分泌代谢杂志

Objective This article reported the clinical characteristics and gene mutations of two pseudoachondroplasia cases, and made a literature review in order to improve clinicians' understanding of the disease. Methods Clinical features of two patients who were short stature accompanied with skeletal deformities were summarized, and they accepted whole exome sequencing. We also reviewed literature to summarize the clinical characteristics and known gene research progress of all reported Chinese pseudoachondroplasia cases. Results The two patients' clinical characteristics were short limbdwarfism with skeletal deformity. Genetic results showed that there were two heterozygous mutations in the cartilage oligomeric matrix protein (COMP) gene of the two patients, c.14171419delGAC and c.1552G>A, respectively. Up to March 2019, a total of 58 cases of pseudoachondroplasia have been reported in China. The median height of these patients is-5.03 SDS. The clinical features include abnormal gait, short limbs, short fingers/toes, scoliosis, bracelet sign, ankle sign and other skeletal deformities. COMP is the pathogenic gene and mutations mainly located in calmodulin-like domains. The hotspot mutation is c. 14171419delGAC. Conclusions Pseudoachondroplasia is a kind of rare genetic disease characterized by short stature and skeletal deformities. The clinical and genetic characteristics of the disease were summarized, which may improve the early diagnosis rate.

Clinical features and gene mutation analysis of COMP in eight patients with pseudoachondroplasia / 中华内分泌代谢杂志

The clinical features and the mutation of cartilage oligomer matrix protein (COMP) gene were analyzed in 8 patients with pseudoachondroplasia (PSACH).The clinical data and the peripheral blood from 5 male and 3 female probands,their pedigree members,and 250 unrelated volunteers were collected.Eight patients who were sporadic cases,had been detected mutation of COMP gene by DNA sequencing.PSACH is a skeletal disorder characterized by short stature,joint laxity,and early-onset osteoarthritis.The heights of 8 patients were significantly lower than the average level by 3 standard deviations,with short limbs and deformities of legs.Radiographs showed flattening of vertebrae with anterior beaking or tonguing in children and osteoarthritis in adults.As to the patients with short limb dwarfism,short toes,and abnormal radiography findings,PSACH should be suspected and could be confirmed by detection of COMP gene mutation.

The effects of leptin on osteogenesis/odontogenic related gene expression of human apical papillary stem cells / 口腔疾病防治

Objective@#To investigate the effects of leptin on the proliferation of stem cells from human stem cells from the apical papilla (hSCAPs) and the expression of osteogenic/dentinogenic genes in vitro to provide an experimental basis for the sustainable development of young permanent teeth. @* Methods @#The tissue block method was used to isolate and culture hSCAPs from the apical papilla of the immature third permanent molar. The expression of leptin and OBRb in hSCAPs was detected using immunocytofluorescence staining, western blotting and reverse transcription polymerase chain reaction (RT-PCR) analysis. The hSCAPs was treated with 0.1 μg/mL of leptin (0.1 μg/mL group) or 1.5 μg/mL of leptin (1.5 μg/mL group) at different time points. The control group was treated with alpha-MEM medium. Cell proliferation was measured using the CCK8 assay and cell cycle analysis. QRT-PCR was used to detect the expression of related osteoblast/odontogenic genes for alkaline phosphatase (ALP), dentin matrix protein -1 (DMP-1), dentin sialophosphoprotein (DSPP), and osteocalcin (OCN) mRNA. The differences between the treatment groups and the control group were analyzed statistically using one-way ANOVA followed by Bonferroni analysis.@*Results@#The expression of both leptin and OBRb were found in hSCAPs. Compared with the control group, the cell proliferation capacity and S phase cells in the treatment groups were higher than those in the control group, with the 1.5 μg /mL group displaying higher levels than 0.1 μg /mL group, and the treated hSCAPs demonstrated a higher proliferation rate and a higher expression of ALP, DSPP, and DMP-1 from day 3 to day 7, with the 1.5 μg /mL group displaying higher levels than 0.1 μg /mL group , and the difference was statistically significant (P < 0.05), at day 7. The treated hSCAPs demonstrated a lower expression of ALP, DSPP, and DMP-1. Compared with the control group, the treated hSCAPs demonstrated a higher expression of OCN from day 7 to day 14, with significantly higher expression in the 1.5 μg /mL group compared to the 0.1 μg /mL group.@*Conclusion@#Leptin may promote cell proliferation and upregulate the expression of relative osteogenic/dentinogenic genes.