Objective:To explored the effect of 78c in treating collagen-induced arthritis (CIA) mice and to investigate its mechanism of effects.Methods:CIA mice model and CD38 +NK cells were treated with 78c. Cytokine concentrations and lymphocyte subtypes were measured in the mice peripheral blood and culture medium using flow cytometry. Mikenyi cell isolation kit was used to isolate CD4 + T cells and NK cells from peripheral blood mononuclear cells of healthy volunteers. CD38 + NK cells were enriched using the Miltenyi CD38 microbeads from the extracted NK cells. CD38 + NK cells with 78c pretreatment or not were cocultured with CD4 +T cells in transwells. The least significant difference (LSD) method was used for comparison between the two groups, and one-way analysis of variance was used for multi-group significance. Pearson correlation analysis was used for correlation analysis. Results:78c treatment significantly suppressed joint inflammation, inhibited the toe thickness of CIA mice, and reduced the number of while cell, neutrophils, platelets, and concentrations of IFN-γ, IL-6 and TNF-α ( t=6.10, P<0.001; t=4.00, P=0.002; t=3.09, P=0.012; t=2.31, P=0.043; t=3.58, P=0.005; t=2.68, P=0.002) in the CIA mice. The proportion of CD38 +NK cells decreased from (3.9±0.9)% to (2.4±0.3)% ( t=2.49, P=0.032), the proportion of regulatory T cell (Treg) increased from (0.81±0.33)% to (1.41±0.26)% ( t=2.74, P=0.021), and the concentration of IL-10 also increased from (99±37) pg/ml to (199±9) pg/ml( t=2.76 , P=0.020). The proportion of Treg in CD4 +T cells cocultured with 78c-pretreated CD38 +NK cells increased from (0.52±0.04)% to (0.69±0.08)% ( t=3.33, P=0.029) , the T helper cells (Th)17/Treg ratio decreased from (4.44±0.26) to (2.59±0.64) ( t=4.76 , P=0.009), and the Th1/Th2 ratio decreased from (14.8±1.6) to (8.1±1.3)( t=5.70 , P=0.005). Conclusion:78c can reduce the proportion of CD38 +NK cells, thereby reducing the inhibition of CD38 +NK cells on CD4 +T cell differentiation into Treg cells, leading to the restoration of immune balance. The results of this study suggest that 78c is a potential therapeutic agent for rheumatoid arthritis.

ObjectiveTo automatically measure the wrist range of motion using the somatosensory capture device Azure Kinect. MethodsAzure Kinect was used to recognize the spatial coordinates of human elbow, wrist, palm, fingertip and other joint points, and the best measurement posture was determined by orthogonal experiment. Holt's double-parameter exponential smoothing method and bone length constraint method were used to smooth the joint point data to eliminate the jitter. The average angle of multiple frames was calculated through the spatial vector relationship to realize the automatic measurement of wrist joint range of motion. From May to October, 2021, 5 times × 10 groups of measurement were performed on each subject of wrist joint of five healthy subjects using the above method. ResultsThe R1 of each subject of wrist joint was between 83 and 127, and the value was in the middle. This method contrasted well with measurements made on augmented reality ruler, 2D images (r > 0.990), and the maximum error was within 1.61°. ConclusionThe wrist joint angle measurement modality using Azure Kinect is consistent to conventional measurement modality, and can meet the requirements of real-time measurement.

Objective: To investigate the effect of siRNA silencing α1 antitrypsin (α1-AT) gene on the biological behavior of rheumatoid arthritis fibroblast-like synoviocytes (RA-SFs). Methods: Primary culture of knee synovial tissue from 5 patients with rheumatoid arthritis (RA) was performed. The artificially synthesized silencing α1-AT siRNA specifically inhibits the expression of α1-AT in RA-SFs. After 24 and 36 hours of transient transfection, the inhibition efficiency was detected by Quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the expression of related genes after α1-AT gene silencing was detected.Furthermore, ethyl thiazolyl tetrazolium (MTT) assay, Trans-well chamber, cell scratch and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of interfering α1-AT expression on cell proliferation, invasion and migration, and secretion of interleukin (IL)-17, Tumor necrosis factor (TNF)-α, IL-1α, IL-1β and other related inflammatory factors. At the same time, when the pathway inhibitor (ERK inhibitor, signal transducer and activator of transcription 3 (STAT3) inhibitor, NF-κB inhibitor) stimulated cells, the effect on α1-AT was changed. One-way analysis of variance was used for comparison between the two groups; further pairwise comparison using LSD-t test; The count data was checked by χ² test. Results: Compared with the negative control group, the siRNA α1-AT group was transfected for 24 hours, the α1-AT mRNA level was significantly inhibited (P<0.05), and the proliferation rate of RA-SFs was not affected (P>0.05), and the invasion and migration ability of cells decreased significantly (P<0.05). The secretion of IL-17 and IL-1β was slightly decreased (P>0.05), while TNF-α and IL-1α were not affected (P>0.05). In addition, the expression of α1-AT downstream genes Matrix metallop roteinases (MMP)-2 169, Hu-Bax1, MMP-9, Hu Bax and Hu Bcl-xl were significantly decreased (P<0.05). Moreover, the signaling pathway inhibitors ERK inhibitor (PD98059) and NF-κB inhibitor (PDTC) had significant effects on α1-AT (P<0.05). Conclusion The α1-AT gene silencing has potential effect on the biological behavior of RA SFs. Further study of this mechanism is helpful to provide evidence for the treatment of RA.

Objective: To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist. Methods: The levels of NK cells, CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3, CCR5), as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid. The isolated cells were then cultured in vitro and divided into 3 groups, namely blank control group, IB4 stimulation group (Anti-CD38 mAb), IL-2 and IB4 co-stimulation group. And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA). Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients, and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR). The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot. Results: The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P<0.05). The two chemokine receptors (CXCR3, CCR5) were mainly expressed in CD38⁺ NK cell subsets. The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P<0.05). The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P<0.05). The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P<0.05). Conclusions The synovial NK cells are more lethal than the peripheral NK cells in the RA patients. CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway. Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins. CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.

The protein encoded by TXNDC5 is a member the protein disulfide isomerase family, which has disulfide isomerase activity and can act as the molecular chaperone to reduce the synthesis of abnormal proteins. Its biological functions include anti-oxidation, promoting angiogenesis, taking part in cellular inflammation, and energy metabolism, etc. Studies have demonstrated that the expression of TXNDC5 is increased in many types of tumors including cervical carcinoma, gastric carcinoma and colorectal cancer. Moreover, TXNDC5 is also closely associated with rheumatoid arthritis, diabetes, hepatic steatosis and vitiligo. This paper aims to summarize the latest progress in research on TXNDC5 in terms of biochemical function, relationship with diseases and the underlying mechanism.
Animals ; Arthritis, Rheumatoid ; enzymology ; genetics ; Diabetes Mellitus ; enzymology ; genetics ; Humans ; Neoplasms ; enzymology ; genetics ; Protein Disulfide-Isomerases ; genetics

OBJECTIVEThe conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. The present study aimed to identify novel citrullinated proteins in 10 tumor cell lines by 2-D Western blotting (2-D WB).

METHODSTwo identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ten cultured human tumor cell lines: ECA(esophageal cancer cells), HEPG2 (hepatocellular carcinoma cells), SKOV3 (ovarian cancer cells), MCF-7 (breast cancer cells), H292 (lung mucoepidermoid carcinoma cells), HeLa (cervical cancer cells), Lovo (colon cancer cells), OS-RC (renal cell carcinoma cells), PANC-1 (pancreatic cancer cells), and SGC (gastric cancer cells). The expression profiles on one 2-DE gels were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in the tumor cell lines.

RESULTS2-D WB and mass spectrometry identified citrullinated ENO1 (α-enolase), HSP60 (heat shock protein 60), KRT8 (keratin 8), TUBB (tubulin beta), TCRβ (T cell receptor β chain), VIME (vimentin) and PDI in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumor cell lines.

CONCLUSIONThe citrullination of proteins ENO1, HSP60, KRT8, and TUBB suggests a new mechanism in the tumorigenic process.

Blotting, Western ; Cell Line, Tumor ; Citrulline ; metabolism ; Female ; Humans ; Immunoprecipitation ; Mass Spectrometry ; Phosphopyruvate Hydratase ; Vimentin

The manuscript introduced the overview, training objectives, policy advantages, training process,curriculum, examination of the Australian College in Rural and Remote Medicine and further contrasted that with China's domestics.The authors held that Australia's training is better targeted due to its colleges tailored to this end;the training duration for general practitioners of rural and remote areas is longer,and the training schedule is reasonable;the curriculum design and training content are more targeted;and the homogeneous training is better achieved as its examination is run by the college in a standardized manner.The authors therefore hold that China should develop detailed regulations for general practitioners from rural and remote areas and explore the feasibility of setting up second-level disciplines institutes for internal medicine, surgery, gynecology and obstetrics, pediatrics and general at national and provincial level.

Objective The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins.The present study aimed to identify novel citrullinated proteins in 10 tumor cell lines by 2-D Western blotting (2-D WB).Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ten cultured human tumor cell lines: ECA ( esophageal cancer cells ) , HEPG2 ( hepatocellular carcinoma cells ) , SKOV3 ( ovarian cancer cells),MCF-7 (breast cancer cells),H292 (lung mucoepidermoid carcinoma cells), HeLa (cervical cancer cells), Lovo (colon cancer cells), OS-RC (renal cell carcinoma cells), PANC-1 (pancreatic cancer cells), and SGC (gastric cancer cells).The expression profiles on one 2-DE gels were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody.By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry.Immunoprecipitation was used to verify the expressionandcitrullinationofthetargetedproteinsinthetumorcelllines.Results 2-DWBandmass spectrometry identified citrullinated ENO1 (α-enolase), HSP60 (heat shock protein 60), KRT8 (keratin 8), TUBB (tubulin beta), TCRβ(T cell receptor βchain), VIME (vimentin) and PDI in these cell lines.Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumor cell lines.Conclusion The citrullination of proteins ENO1, HSP60, KRT8, and TUBB suggests a new mechanism in the tumorigenic process.

Objective The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins.The present study aimed to identify novel citrullinated proteins in 10 tumor cell lines by 2-D Western blotting (2-D WB).Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ten cultured human tumor cell lines: ECA ( esophageal cancer cells ) , HEPG2 ( hepatocellular carcinoma cells ) , SKOV3 ( ovarian cancer cells),MCF-7 (breast cancer cells),H292 (lung mucoepidermoid carcinoma cells), HeLa (cervical cancer cells), Lovo (colon cancer cells), OS-RC (renal cell carcinoma cells), PANC-1 (pancreatic cancer cells), and SGC (gastric cancer cells).The expression profiles on one 2-DE gels were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody.By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry.Immunoprecipitation was used to verify the expressionandcitrullinationofthetargetedproteinsinthetumorcelllines.Results 2-DWBandmass spectrometry identified citrullinated ENO1 (α-enolase), HSP60 (heat shock protein 60), KRT8 (keratin 8), TUBB (tubulin beta), TCRβ(T cell receptor βchain), VIME (vimentin) and PDI in these cell lines.Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumor cell lines.Conclusion The citrullination of proteins ENO1, HSP60, KRT8, and TUBB suggests a new mechanism in the tumorigenic process.

Objective To observe the inhibiting activities of fibronectin (FN) and citrullinated fibronectin (cFN) on disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4),and to explore the extracellular regulative mechanisms of ADAMTS-4.Methods FN was incubated with peptidylarginine deaminase type 4 (PADI4).Western blotting analysis was used to verify the citrullination of FN.The binding activity of FN and cFN to ADAMTS-4 were investigated by enzyme-linked immunosorbent assay (ELISA).The proteolytic ability of ADAMTS-4 after binding to FN and cFN were measured with the aggrecanase activity assay kit.One-way ANOVA,LSD-t test and t-test were used for statistical analysis.Results The immunosignal of citrulline was detected in FN after incubated with PADI4,but not in the absence of PADI4.A higher absorbance at 405 nm was detected when the full-length ADAMTS-4 protein was incubated with FN (2.182±0.042) than cFN (0.624±0.033; t=50.522,P＜0.01).Additionally,the recombinant ADAMTS-4 protein with a truncation at the C-terminus displayed low absorbance at 405 nm when the enzyme was incubated with both FN(0.971±0.024) and cFN(0.934±0.012; t=2.388,P＞0.05).Large amounts of ARGxx peptide were detected with full-length ADAMTS-4 in aggrecanase activity assay [(0.908±0.088) nmol/L],but significantly less when in the presence of FN and ADAMTS-4 [(0.573±0.000) nmol/L,P＜0.05].The production of this peptide was more when full-length ADAMTS-4 was incubated with cFN [(0.830±0.020) nmol/L,P＜0.05] than with FN.The reaction containing the truncated ADAMTS-4 without FN or cFN yielded the highest concentration of ARGxx peptide [(36.420±3.673) nmol/L],peptide production was not significantly altered when FN [(41.099±0.101) nmol/L] or eFN [(41.064±0.083) nmol/L] were added to the reaction.Conclusion FN could bind to ADAMTS-4 and inhibit its proteolytic activity.After citrullinated by PADI4,the binding activity of cFN is weakened and less inhibition to ADAMTS-4.